RB regulates the production and the survival of newborn neurons in the embryonic and adult dentate gyrus.

In mammals, hippocampal dentate gyrus granule cells (DGCs) constitute a particular neuronal population produced both during embryogenesis and adult life, and play key roles in neural plasticity and memory. However, the molecular mechanisms regulating neurogenesis in the dentate lineage throughout development and adulthood are still not well understood. The Retinoblastoma protein (RB), a transcriptional repressor primarily involved in cell cycle control and cell death, plays crucial roles during cortical development but its function in the formation and maintenance of DGCs remains unknown. Here, we show that loss of RB during embryogenesis induces massive ectopic proliferation and delayed cell cycle exit of young DGCs specifically at late developmental stages but without affecting stem cells. This phenotype was partially counterbalanced by increased cell death. Similarly, during adulthood, loss of RB causes ectopic proliferation of newborn DGCs and dramatically impairs their survival. These results demonstrate a crucial role for RB in the generation and the survival of DGCs in the embryonic and the adult brain. © 2016 Wiley Periodicals, Inc.

The integrated stress response promotes neural stem cell survival under conditions of mitochondrial dysfunction in neurodegeneration.

Impaired mitochondrial function is a hallmark of aging and a major contributor to neurodegenerative diseases. We have shown that disrupted mitochondrial dynamics typically found in aging alters the fate of neural stem cells (NSCs) leading to impairments in learning and memory. At present, little is known regarding the mechanisms by which neural stem and progenitor cells survive and adapt to mitochondrial dysfunction. Using Opa1-inducible knockout as a model of aging and neurodegeneration, we identify a decline in neurogenesis due to impaired stem cell activation and progenitor proliferation, which can be rescued by the mitigation of oxidative stress through hypoxia. Through sc-RNA-seq, we identify the ATF4 pathway as a critical mechanism underlying cellular adaptation to metabolic stress. ATF4 knockdown in Opa1-deficient NSCs accelerates cell death, while the increased expression of ATF4 enhances proliferation and survival. Using a Slc7a11 mutant, an ATF4 target, we show that ATF4-mediated glutathione production plays a critical role in maintaining NSC survival and function under stress conditions. Together, we show that the activation of the integrated stress response (ISR) pathway enables NSCs to adapt to metabolic stress due to mitochondrial dysfunction and metabolic stress and may serve as a therapeutic target to enhance NSC survival and function in aging and neurodegeneration.

Early postnatal defects in neurogenesis in the 3xTg mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder leading to dementia. The hippocampus, which is one of the sites where neural stem cells reside and new neurons are born, exhibits the most significant neuronal loss in AD. A decline in adult neurogenesis has been described in several animal models of AD. However, the age at which this defect first appears remains unknown. To determine at which stage, from birth to adulthood, the neurogenic deficits are found in AD, we used the triple transgenic mouse model of AD (3xTg). We show that defects in neurogenesis are present as early as postnatal stages, well before the onset of any neuropathology or behavioral deficits. We also show that 3xTg mice have significantly fewer neural stem/progenitor cells, with reduced proliferation and decreased numbers of newborn neurons at postnatal stages, consistent with reduced volumes of hippocampal structures. To determine whether there are early changes in the molecular signatures of neural stem/progenitor cells, we perform bulk RNA-seq on cells sorted directly from the hippocampus. We show significant changes in the gene expression profiles at one month of age, including genes of the Notch and Wnt pathways. These findings reveal impairments in neurogenesis very early in the 3xTg AD model, which provides new opportunities for early diagnosis and therapeutic interventions to prevent neurodegeneration in AD.

Direct FACS Isolation of Neural Stem/Progenitor Lineages from the Adult Brain.

Adult neural stem and progenitor cells reside in the neurogenic niche of the adult brain and have tremendous potential in regenerative medicine. Compelling evidence suggests that adult neurogenesis plays an important role in hippocampal memory formation, plasticity, and mood regulation. Understanding the mechanisms that regulate the function of neural stem/progenitor cells within the brain is a critical step for the development of regenerative strategies to maintain or enhance neurological function. A major challenge in studying these cells is the limited cell number of adult neural stem cells, and the significant changes in their properties induced by in vitro culture and expansion. To best understand the regulation of these cells, they must be studied within their niche context. In this chapter, we provide a simplified protocol for the harvest and isolation of neural stem cell lineages directly from the murine brain, to provide input material for single-cell RNA-seq. This approach will elucidate the true transcriptional signatures and activated pathways in neural stem cell lineages, within the context of their niche environment.

The Rb/E2F axis is a key regulator of the molecular signatures instructing the quiescent and activated adult neural stem cell state.

Long-term maintenance of the adult neurogenic niche depends on proper regulation of entry and exit from quiescence. Neural stem cell (NSC) transition from quiescence to activation is a complex process requiring precise cell-cycle control coordinated with transcriptional and morphological changes. How NSC fate transitions in coordination with the cell-cycle machinery remains poorly understood. Here we show that the Rb/E2F axis functions by linking the cell-cycle machinery to pivotal regulators of NSC fate. Deletion of Rb family proteins results in activation of NSCs, inducing a transcriptomic transition toward activation. Deletion of their target activator E2Fs1/3 results in intractable quiescence and cessation of neurogenesis. We show that the Rb/E2F axis mediates these fate transitions through regulation of factors essential for NSC function, including REST and ASCL1. Thus, the Rb/E2F axis is an important regulator of NSC fate, coordinating cell-cycle control with NSC activation and quiescence fate transitions.

RB: An essential player in adult neurogenesis.

The fundamental mechanisms underlying adult neurogenesis remain to be fully clarified. Members of the cell cycle machinery have demonstrated key roles in regulating adult neural stem cell (NSC) quiescence and the size of the adult-born neuronal population. The retinoblastoma protein, Rb, is known to possess CNS-specific requirements that are independent from its classical role as a tumor suppressor. The recent study by Vandenbosch et al. has clarified distinct requirements for Rb during adult neurogenesis, in the restriction of proliferation, as well as long-term adult-born neuronal survival. However, Rb is no longer believed to be the main cell cycle regulator maintaining the quiescence of adult NSCs. Future studies must consider Rb as part of a larger network of regulatory effectors, including the other members of the Rb family, p107 and p130. This will help elucidate the contribution of Rb and other pocket proteins in the context of adult neurogenesis, and define its crucial role in regulating the size and fate of the neurogenic niche.