Microglia Actively Remodel Adult Hippocampal Neurogenesis through the Phagocytosis Secretome

During adult hippocampal neurogenesis, most newborn cells undergo apoptosis and are rapidly phagocytosed by resident microglia to prevent the spillover of intracellular contents. Here, we propose that phagocytosis is not merely passive corpse removal but has an active role in maintaining neurogenesis. During adult hippocampal neurogenesis, most newborn cells undergo apoptosis and are rapidly phagocytosed by resident microglia to prevent the spillover of intracellular contents. Here, we propose that phagocytosis is not merely passive corpse removal but has an active role in maintaining neurogenesis. First, we found that neurogenesis was disrupted in male and female mice chronically deficient for two phagocytosis pathways: the purinergic receptor P2Y12, and the tyrosine kinases of the TAM family Mer tyrosine kinase (MerTK)/Axl. In contrast, neurogenesis was transiently increased in mice in which MerTK expression was conditionally downregulated. Next, we performed a transcriptomic analysis of the changes induced by phagocytosis in microglia in vitro and identified genes involved in metabolism, chromatin remodeling, and neurogenesis-related functions. Finally, we discovered that the secretome of phagocytic microglia limits the production of new neurons both in vivo and in vitro. Our data suggest that microglia act as a sensor of local cell death, modulating the balance between proliferation and survival in the neurogenic niche through the phagocytosis secretome, thereby supporting the long-term maintenance of adult hippocampal neurogenesis.  Microglia are the brain professional phagocytes and, in the adult hippocampal neurogenic niche, they remove newborn cells naturally undergoing apoptosis. Here we show that phagocytosis of apoptotic cells triggers a coordinated transcriptional program that alters their secretome, limiting neurogenesis both in vivo and in vitro. In addition, chronic phagocytosis disruption in mice deficient for receptors P2Y12 and MerTK/Axl reduces adult hippocampal neurogenesis. In contrast, inducible MerTK downregulation transiently increases neurogenesis, suggesting that microglial phagocytosis provides a negative feedback loop that is necessary for the long-term maintenance of adult hippocampal neurogenesis. Therefore, we speculate that the effects of promoting engulfment/degradation of cell debris may go beyond merely removing corpses to actively promoting regeneration in development, aging, and neurodegenerative diseases.

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abstract: Microglia are primary immune sentinels of the CNS. Following injury, these cells migrate or extend processes toward sites of tissue damage. CNS injury is accompanied by release of nucleotides, serving as signals for microglial activation or chemotaxis. Microglia express several purinoceptors, including a Gi-coupled subtype that has been implicated in ATP- and ADP-mediated migration in vitro. Here we show that microglia from mice lacking Gi-coupled P2Y12 receptors exhibit normal baseline motility but are unable to polarize, migrate or extend processes toward nucleotides in vitro or in vivo. Microglia in P2ry12−/− mice show significantly diminished directional branch extension toward sites of cortical damage in the living mouse. Moreover, P2Y12 expression is robust in the 'resting' state, but dramatically reduced after microglial activation. These results imply that P2Y12 is a primary site at which nucleotides act to induce microglial chemotaxis at early stages of the response to local CNS injury.

title: Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance
abstract: Phagocytic removal of apoptotic cells occurs efficiently in vivo such that even in tissues with significant apoptosis, very few apoptotic cells are detectable. This is thought to be due to the release of ‘find-me’ signals by apoptotic cells that recruit motile phagocytes such as monocytes, macrophages and dendritic cells, leading to the prompt clearance of the dying cells. However, the identity and in vivo relevance of such find-me signals are not well understood. Here, through several lines of evidence, we identify extracellular nucleotides as a critical apoptotic cell find-me signal. We demonstrate the caspase-dependent release of ATP and UTP (in equimolar quantities) during the early stages of apoptosis by primary thymocytes and cell lines. Purified nucleotides at these concentrations were sufficient to induce monocyte recruitment comparable to that of apoptotic cell supernatants. Enzymatic removal of ATP and UTP (by apyrase or the expression of ectopic CD39) abrogated the ability of apoptotic cell supernatants to recruit monocytes in vitro and in vivo. We then identified the ATP/UTP receptor P2Y2 as a critical sensor of nucleotides released by apoptotic cells using RNA interference-mediated depletion studies in monocytes, and macrophages from P2Y2-null mice. The relevance of nucleotides in apoptotic cell clearance in vivo was revealed by two approaches. First, in a murine air-pouch model, apoptotic cell supernatants induced a threefold greater recruitment of monocytes and macrophages than supernatants from healthy cells did; this recruitment was abolished by depletion of nucleotides and was significantly decreased in P2Y2-/- (also known as P2ry2-/-) mice. Second, clearance of apoptotic thymocytes was significantly impaired by either depletion of nucleotides or interference with P2Y receptor function (by pharmacological inhibition or in P2Y2-/- mice). These results identify nucleotides as a critical find-me cue released by apoptotic cells to promote P2Y2-dependent recruitment of phagocytes, and provide evidence for a clear relationship between a find-me signal and efficient corpse clearance in vivo.

title: The Ki‐67 protein: From the known and the unknown
abstract: The expression of the human Ki‐67 protein is strictly associated with cell proliferation. During interphase, the antigen can be exclusively detected within the nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. The fact that the Ki‐67 protein is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent from resting cells (G0), makes it an excellent marker for determining the so‐called growth fraction of a given cell population. In the first part of this study, the term proliferation marker is discussed and examples of the applications of anti‐Ki‐67 protein antibodies in diagnostics of human tumors are given. The fraction of Ki‐67‐positive tumor cells (the Ki‐67 labeling index) is often correlated with the clinical course of the disease. The best‐studied examples in this context are carcinomas of the prostate and the breast. For these types of tumors, the prognostic value for survival and tumor recurrence has repeatedly been proven in uni‐ and multivariate analysis. The preparation of new monoclonal antibodies that react with the Ki‐67 equivalent protein from rodents now extends the use of the Ki‐67 protein as a proliferation marker to laboratory animals that are routinely used in basic research. The second part of this review focuses on the biology of the Ki‐67 protein. Our current knowledge of the Ki‐67 gene and protein structure, mRNA splicing, expression, and cellular localization during the cell‐division cycle is summarized and discussed. Although the Ki‐67 protein is well characterized on the molecular level and extensively used as a proliferation marker, the functional significance still remains unclear. There are indications, however, that Ki‐67 protein expression is an absolute requirement for progression through the cell‐division cycle.  2000.

title: TAM receptor signaling and autoimmune disease.
abstract: The TAM receptor tyrosine kinases Tyro3, Axl, and Mer and their ligands Gas6 and Protein S are essential for the phagocytosis of apoptotic cells and membranes in the adult immune, nervous, and reproductive systems. Genetic studies indicate that this receptor-ligand system is central to apoptotic cell engulfment that is triggered by the 'eat-me' signal phosphatidylserine (PtdSer). At the same time, TAM signaling is normally activated by Toll-like receptor (TLR) and type I interferon signaling, as part of the innate inflammatory response in dendritic cells (DCs) and macrophages, where it inhibits this response. Deficiencies in TAM signaling result in human retinal dystrophies and may contribute to lupus and other human autoimmune diseases.

title: CX3CR1 Deficiency Leads to Impairment of Hippocampal Cognitive Function and Synaptic Plasticity
abstract: The protective/neurotoxic role of fractalkine (CX3CL1) and its receptor CX3C chemokine receptor 1 (CX3CR1) signaling in neurodegenerative disease is an intricate and highly debated research topic and it is becoming even more complicated as new studies reveal discordant results. It appears that the CX3CL1/CX3CR1 axis plays a direct role in neurodegeneration and/or neuroprotection depending on the CNS insult. However, all the above studies focused on the role of CX3CL1/CX3CR1 signaling in pathological conditions, ignoring the relevance of CX3CL1/CX3CR1 signaling under physiological conditions. No approach to date has been taken to decipher the significance of defects in CX3CL1/CX3CR1 signaling in physiological condition. In the present study we used CX3CR1−/−, CX3CR1+/−, and wild-type mice to investigate the physiological role of CX3CR1 receptor in cognition and synaptic plasticity. Our results demonstrate for the first time that mice lacking the CX3CR1 receptor show contextual fear conditioning and Morris water maze deficits. CX3CR1 deficiency also affects motor learning. Importantly, mice lacking the receptor have a significant impairment in long-term potentiation (LTP). Infusion with IL-1β receptor antagonist significantly reversed the deficit in cognitive function and impairment in LTP. Our results reveal that under physiological conditions, disruption in CX3CL1 signaling will lead to impairment in cognitive function and synaptic plasticity via increased action of IL-1β.

title: Efferocytosis induces a novel SLC program to promote glucose uptake and lactate release
abstract: Development and routine tissue homeostasis require a high turnover of apoptotic cells. These cells are removed by professional and non-professional phagocytes via efferocytosis1. How a phagocyte maintains its homeostasis while coordinating corpse uptake, processing ingested materials and secreting anti-inflammatory mediators is incompletely understood1,2. Here, using RNA sequencing to characterize the transcriptional program of phagocytes actively engulfing apoptotic cells, we identify a genetic signature involving 33 members of the solute carrier (SLC) family of membrane transport proteins, in which expression is specifically modulated during efferocytosis, but not during antibody-mediated phagocytosis. We assessed the functional relevance of these SLCs in efferocytic phagocytes and observed a robust induction of an aerobic glycolysis program, initiated by SLC2A1-mediated glucose uptake, with concurrent suppression of the oxidative phosphorylation program. The different steps of phagocytosis2—that is, ‘smell’ (‘find-me’ signals or sensing factors released by apoptotic cells), ‘taste’ (phagocyte–apoptotic cell contact) and ‘ingestion’ (corpse internalization)—activated distinct and overlapping sets of genes, including several SLC genes, to promote glycolysis. SLC16A1 was upregulated after corpse uptake, increasing the release of lactate, a natural by-product of aerobic glycolysis3. Whereas glycolysis within phagocytes contributed to actin polymerization and the continued uptake of corpses, lactate released via SLC16A1 promoted the establishment of an anti-inflammatory tissue environment. Collectively, these data reveal a SLC program that is activated during efferocytosis, identify a previously unknown reliance on aerobic glycolysis during apoptotic cell uptake and show that glycolytic by-products of efferocytosis can influence surrounding cells.Distinct transcriptional programs are activated during different stages of apoptotic cell engulfment, including a unique program of genes coding for solute carrier proteins and enzymes in the glycolytic pathway.

title: The Mammalian Adult Neurogenesis Gene Ontology (MANGO) Provides a Structural Framework for Published Information on Genes Regulating Adult Hippocampal Neurogenesis
abstract: Adult hippocampal neurogenesis is not a single phenotype, but consists of a number of sub-processes, each of which is under complex genetic control. Interpretation of gene expression studies using existing resources often does not lead to results that address the interrelatedness of these processes. Formal structure, such as provided by ontologies, is essential in any field for comprehensive interpretation of existing knowledge but, until now, such a structure has been lacking for adult neurogenesis.  We have created a resource with three components 1. A structured ontology describing the key stages in the development of adult hippocampal neural stem cells into functional granule cell neurons. 2. A comprehensive survey of the literature to annotate the results of all published reports on gene function in adult hippocampal neurogenesis (257 manuscripts covering 228 genes) to the appropriate terms in our ontology. 3. An easy-to-use searchable interface to the resulting database made freely available online. The manuscript presents an overview of the database highlighting global trends such as the current bias towards research on early proliferative stages, and an example gene set enrichment analysis. A limitation of the resource is the current scope of the literature which, however, is growing by around 100 publications per year. With the ontology and database in place, new findings can be rapidly annotated and regular updates of the database will be made publicly available.  The resource we present allows relevant interpretation of gene expression screens in terms of defined stages of postnatal neuronal development. Annotation of genes by hand from the adult neurogenesis literature ensures the data are directly applicable to the system under study. We believe this approach could also serve as an example to other fields in a ‘bottom-up’ community effort complementing the already successful ‘top-down’ approach of the Gene Ontology.

title: Neural stem cell deforestation as the main force driving the age-related decline in adult hippocampal neurogenesis
abstract: Newborn neurons derived from radial glia-like stem cells located in the dentate gyrus integrate into the adult hippocampal circuitry and participate in memory formation, spatial learning, pattern separation, fear conditioning, and anxiety. This process takes place throughout the life span of mammals, including humans; however, it follows a sharp declining curve. New neurons are generated abundantly during youth but very scarcely in the aged brain. The absolute number of newly generated neurons, or neurogenic output, is determined at different levels along the neurogenic cascade: the activation of quiescent stem cells; the mitotic potential of proliferating precursors; and the survival of neuronal fate-committed precursors. A continuous depletion of the hippocampal neural stem cell pool has been recently proposed as the main force underlying the age-related decline of neurogenesis, in contrast to the previous view of population of neural stem cells whose number remains constant but loses its ability to bear fruit. Nevertheless, the diminished neurogenic output may be reflecting other phenomena such as decreased mitotic capability of proliferating progenitors, decreased survival or changes in differentiation. We describe herein the most important events in determining the amount of neurogenesis in the dentate gyrus and examine the literature to understand the effects of age throughout the cascade.

title: Development and maintenance of the brain's immune toolkit: Microglia and non‐parenchymal brain macrophages
abstract: Microglia and non‐parenchymal macrophages located in the perivascular space, the meninges and the choroid plexus are independent immune populations that play vital roles in brain development, homeostasis, and tissue healing. Resident macrophages account for a significant proportion of cells in the brain and their density remains stable throughout the lifespan thanks to constant turnover. Microglia develop from yolk sac progenitors, later evolving through intermediate progenitors in a fine‐tuned process in which intrinsic factors and external stimuli combine to progressively sculpt their cell type‐specific transcriptional profiles. Recent evidence demonstrates that non‐parenchymal macrophages are also generated during early embryonic development. In recent years, the development of powerful fate mapping approaches combined with novel genomic and transcriptomic methodologies have greatly expanded our understanding of how brain macrophages develop and acquire specialized functions, and how cell population dynamics are regulated. Here, we review the transcription factors, epigenetic remodeling, and signaling pathways orchestrating the embryonic development of microglia and non‐parenchymal macrophages. Next, we describe the dynamics of the macrophage populations of the brain and discuss the role of progenitor cells, to gain a better understanding of their functions in the healthy and diseased brain.

title: Transient expression of doublecortin during adult neurogenesis
abstract: During development of the central nervous system, expression of the microtubule binding protein doublecortin (DCX) is associated with migration of neuroblasts. In addition to this developmental role, expression of DCX remains high within certain areas of the adult mammalian brain. These areas, mainly the dentate gyrus and the lateral ventricle wall in conjunction with the rostral migratory stream and olfactory bulb, retain the capacity to generate new neurons into adulthood. Adult neurogenesis is typically detected by incorporation of bromodeoxyuridine (BrdU) into dividing cells and colabeling of BrdU‐positive cells with markers for mature neurons. To elucidate whether DCX could act as an alternative indicator for adult neurogenesis, we investigated the temporal expression pattern of DCX in neurogenic regions of the adult brain. Analysis of newly generated cells showed that DCX is transiently expressed in proliferating progenitor cells and newly generated neuroblasts. As the newly generated cells began expressing mature neuronal markers, DCX immunoreactivity decreased sharply below the level of detection and remained undetectable thereafter. The transient expression pattern of DCX in neuronal committed progenitor cells/neuroblasts indicates that DCX could be developed into a suitable marker for adult neurogenesis and may provide an alternative to BrdU labeling. This assumption is further supported by our observation that the number of DCX‐expressing cells in the dentate gyrus was decreased with age according to the reduction of neurogenesis in the aging dentate gyrus previously reported.

title: Spatial and temporal expression of S100B in cells of oligodendrocyte lineage
abstract: The analysis of oligodendrocyte (OL) lineage development has been facilitated by the immunocytochemical characterization of OL‐specific antigens and definition of the phenotypes sequentially acquired by differentiating OLs. The purpose of the present study was to address an enduring discrepancy between several reported cases of S100B immunodetection in CNS myelin and myelinating OLs on the one hand, and the systematic use of the S100B protein as an alleged astrocytic marker in studies of the mammalian CNS on the other. To resolve this discrepancy, we have compared the developmental distribution of EGFP+ cells in the CNS of s100b‐enhanced green fluorescent protein (EGFP) (Vives et al., 2003) and cnp‐EGFP (Yuan et al., 2002) mice, and examined the degree of overlap between EGFP expression and that of stage‐specific markers of OL differentiation during the embryonic and postnatal phases of development. We demonstrate that the S100B protein is expressed in postnatal and adult populations of NG2+ progenitors of mouse brain, as well as in immature and mature myelinating OLs present in the brain and spinal cord of embryonic and adult mice, respectively. Comparison between EGFP and endogenous S100B expression in the s100b‐EGFP and cnp‐EGFP mice indicates that S100B protein expression is upregulated in immature and mature OLs. These results argue against the current view that S100B expression is restricted to the astrocytic lineage in the CNS, and indicate that the use of S100B in combination with other molecular markers will help discriminate oligodendrocytes from astrocytes.

title: Functional Identification of Cell Phenotypes Differentiating from Mice Retinal Neurospheres Using Single Cell Calcium Imaging
abstract: Degeneration of neural retina causes vision impairment and can lead to blindness. Neural stem and progenitor cells might be used as a tool directed to regenerative medicine of the retina. Here, we describe a novel platform for cell phenotype-specific drug discovery and screening of proneurogenic factors, able to boost differentiation of neural retinal progenitor cells. By using single cell calcium imaging (SCCI) and a rational-based stimulation protocol, a diversity of cells emerging from differentiated retinal neurosphere cultures were identified. Exposure of retinal progenitor cultures to KCl or to α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) stimulated Ca2+ transients in microtubule-associated protein 2 (MAP-2) positive neurons. Doublecortin (DCX) and polysialated neural cell adhesion molecule (PSA-NCAM) positive neuroblasts were distinguished from differentiated neurons on the basis of their response to muscimol. Ca2+ fluxes in glial fibrillary acidic protein (GFAP) or glutamine synthetase (GS) positive cells were induced by ATP. To validate the platform, neurospheres were treated with brain-derived neurotrophic factor (BDNF) (proneurogenic) or ciliary neurotrophic factor (CNTF) (gliogenic factor). BDNF increased the percentage of differentiated cells expressing Tuj-1 sensitive to KCl or AMPA and reduced the population of cells responding to muscimol. CNTF exposure resulted in a higher number of cells expressing GFAP responding to ATP. All together, our data may open new perspectives for cell type-specific discovery of drug targets and screening of novel proneurogenic factors to boost differentiation of neural retina cells to treat degenerative retinal diseases.

title: Functional evaluation of neural stem cell differentiation by single cell calcium imaging.
abstract: Neurogenesis in the adult mammalian brain occurs in two specific brain areas, the subventricular zone (SVZ) bordering the lateral ventricles and the subgranular zone (SGZ) of the hippocampus. Although these regions are prone to produce new neurons, cultured cells from these neurogenic niches tend to be mixed cultures, containing both neurons and glial cells. Several reports highlight the potential of the self-healing capacity of the brain following injury. Even though much knowledge has been produced on the neurogenesis itself, brain repairing strategies are still far away from patients cure. Here we review general concepts in the neurogenesis field, also addressing the methods available to study neural stem cell differentiation. A major problem faced by research groups and companies dedicated to brain regenerative medicine resides on the lack of good methods to functionally identify neural stem cell differentiation and novel drug targets. To address this issue, we developed a unique single cell calcium imaging-based method to functionally discriminate different cell types derived from SVZ neural stem cell cultures. The unique functional profile of each SVZ cell type was correlated at the single cell level with the immunodetection of specific phenotypic markers. This platform was raised on the basis of the functional response of neurons, oligodendrocytes and immature cells to depolarising agents, to thrombin and to histamine, respectively. We also outline key studies in which our new platform was extremely relevant in the context of drug discovery and development in the area of brain regenerative medicine.

title: BMP-induced REST regulates the establishment and maintenance of astrocytic identity
abstract: Astrocyte differentiation and maintenance is promoted by BMP signaling, which induces REST/NRSF to repress neuronal genes.

title: Ascl1 Converts Dorsal Midbrain Astrocytes into Functional Neurons In Vivo
abstract: In vivo induction of non-neuronal cells into neurons by transcription factors offers potential therapeutic approaches for neural regeneration. Although generation of induced neuronal (iN) cells in vitro and in vivo has been reported, whether iN cells can be fully integrated into existing circuits remains unclear. Here we show that expression of achaete-scute complex homolog-like 1 (Ascl1) alone is sufficient to convert dorsal midbrain astrocytes of mice into functional iN cells in vitro and in vivo. Specific expression of Ascl1 in astrocytes by infection with GFAP–adeno-associated virus (AAV) vector converts astrocytes in dorsal midbrain, striatum, and somatosensory cortex of postnatal and adult mice into functional neurons in vivo. These iN cells mature progressively, exhibiting neuronal morphology and markers, action potentials, and synaptic inputs from and output to existing neurons. Thus, a single transcription factor, Ascl1, is sufficient to convert brain astrocytes into functional neurons, and GFAP–AAV is an efficient vector for generating iN cells from astrocytes in vivo.

title: In Vivo Clonal Analysis Reveals Self-Renewing and Multipotent Adult Neural Stem Cell Characteristics
abstract: Neurogenesis and gliogenesis continue in discrete regions of the adult mammalian brain. A fundamental question remains whether cell genesis occurs from distinct lineage-restricted progenitors or from self-renewing and multipotent neural stem cells in the adult brain. Here, we developed a genetic marking strategy for lineage tracing of individual, quiescent, and nestin-expressing radial glia-like (RGL) precursors in the adult mouse dentate gyrus. Clonal analysis identified multiple modes of RGL activation, including asymmetric and symmetric self-renewal. Long-term lineage tracing in vivo revealed a significant percentage of clones that contained RGL(s), neurons, and astrocytes, indicating capacity of individual RGLs for both self-renewal and multilineage differentiation. Furthermore, conditional Pten deletion in RGLs initially promotes their activation and symmetric self-renewal but ultimately leads to terminal astrocytic differentiation and RGL depletion in the adult hippocampus. Our study identifies RGLs as self-renewing and multipotent neural stem cells and provides novel insights into in vivo properties of adult neural stem cells.

title: Division-coupled astrocytic differentiation and age-related depletion of neural stem cells in the adult hippocampus.
abstract: Production of new neurons in the adult hippocampus decreases with age; this decline may underlie age-related cognitive impairment. Here we show that continuous depletion of the neural stem cell pool, as a consequence of their division, may contribute to the age-related decrease in hippocampal neurogenesis. Our results indicate that adult hippocampal stem cells, upon exiting their quiescent state, rapidly undergo a series of asymmetric divisions to produce dividing progeny destined to become neurons and subsequently convert into mature astrocytes. Thus, the decrease in the number of neural stem cells is a division-coupled process and is directly related to their production of new neurons. We present a scheme of the neurogenesis cascade in the adult hippocampus that includes a proposed "disposable stem cell" model and accounts for the disappearance of hippocampal neural stem cells, the appearance of new astrocytes, and the age-related decline in the production of new neurons.

title: The Intestinal Stem Cell Niche: Homeostasis and Adaptations.
abstract: The intestinal epithelium is a rapidly renewing cellular compartment. This constant regeneration is a hallmark of intestinal homeostasis and requires a tightly regulated balance between intestinal stem cell (ISC) proliferation and differentiation. Since intestinal epithelial cells directly contact pathogenic environmental factors that continuously challenge their integrity, ISCs must also actively divide to facilitate regeneration and repair. Understanding niche adaptations that maintain ISC activity during homeostatic renewal and injury-induced intestinal regeneration is therefore a major and ongoing focus for stem cell biology. Here, we review recent concepts and propose an active interconversion of the ISC niche between homeostasis and injury-adaptive states that is superimposed upon an equally dynamic equilibrium between active and reserve ISC populations.

title: Neuronal hyperactivity accelerates depletion of neural stem cells and impairs hippocampal neurogenesis.
abstract: Adult hippocampal neurogenesis is believed to maintain a range of cognitive functions, many of which decline with age. We recently reported that radial neural stem cells (rNSCs) in the hippocampus undergo activation-dependent conversion into astrocytes, a mechanism that over time contributes to a reduction in the rNSC population. Here, we injected low and high levels of kainic acid (KA) in the dentate gyrus to assess whether neuronal hyperexcitation, a hallmark of epileptic disorders, could accelerate this conversion. At low levels of KA, generating epileptiform activity without seizures, we indeed found increased rNSC activation and conversion into astrocytes. At high levels, generating sustained epileptic seizures, however, we find that rNSCs divide symmetrically and that both mother and daughter cells convert into reactive astrocytes. Our results demonstrate that a threshold response for neuronal hyperexcitation provokes a dramatic shift in rNSC function, which impairs adult hippocampal neurogenesis in the long term.

title: CD169+ macrophages provide a niche promoting erythropoiesis under homeostasis and stress
abstract: A role for macrophages in erythropoiesis was suggested several decades ago when erythroblastic islands in the bone marrow, composed of a central macrophage surrounded by developing erythroblasts, were described. However, the in vivo role of macrophages in erythropoiesis under homeostatic conditions or in disease remains unclear. We found that specific depletion of CD169+ macrophages markedly reduced the number of erythroblasts in the bone marrow but did not result in overt anemia under homeostatic conditions, probably because of concomitant alterations in red blood cell clearance. However, CD169+ macrophage depletion significantly impaired erythropoietic recovery from hemolytic anemia, acute blood loss and myeloablation. Furthermore, macrophage depletion normalized the erythroid compartment in a JAK2V617F-driven mouse model of polycythemia vera, suggesting that erythropoiesis in polycythemia vera remains under the control of macrophages in the bone marrow and splenic microenvironments. These results indicate that CD169+ macrophages promote late erythroid maturation and that modulation of the macrophage compartment may be a new strategy to treat erythropoietic disorders.

title: Notch ligand Dll1 mediates cross-talk between mammary stem cells and the macrophageal niche
abstract: Cross-talk in the mammary gland Macrophages engulf damaged and dead cells to clear infection, but they also participate in tissue regeneration. Chakrabarti et al. expand the macrophage repertoire for mammary gland development (see the Perspective by Kannan and Eaves). Mammary gland stem cells secrete the Notch ligand Dll1 and activate Notch signaling, which promotes survival of adjacent macrophages. This stimulates production of Wnt ligands, which signal back to the mammary gland stem cells. This cross-talk plays an important role in coordinating mammary gland development, tissue homeostasis, and, not least, breast cancer. Science, this issue p. ; see also p. 1401 Cross-talk between mammary stem cells and macrophages involves Notch and Wnt to regulate mammary development and function.  The stem cell niche plays a crucial role in regulating key stem cell properties, including self-renewal, differentiation, and cell fate change. Although stem cell niches in several organs have been well described, the cellular and molecular characteristics of the mammary gland stem cell (MaSC) niche remain largely underexplored. Stromal cell populations—including fibroblasts, macrophages, and other immune cells—are important for mammary gland development and have been implicated in MaSC niche function. However, the signaling mechanisms driving mammary stromal cell–dependent regulation of MaSC activity remain elusive. Insight into the cross-talk between MaSCs and the niche cells is important for understanding both normal tissue homeostasis and disease conditions such as breast cancer.  Notch signaling is broadly involved in cell fate regulation during development. Although Notch receptors have been implicated in various aspects of mammary gland development, the role of Notch ligands in MaSC regulation is less clear. In this study, we focus on the Notch ligand Dll1, which is highly expressed in MaSC-enriched mammary epithelial cell (MEC) populations. Conditional knockout (cKO) of Dll1 in MECs resulted in a significant delay in branching morphogenesis during mammary gland development and a deficiency in alveoli formation during pregnancy and lactation, suggesting a key role of Dll1-mediated pathways in mammary gland development.  We found that Dll1cKO mice have a reduced number of MaSCs at different stages of mammary gland development in virgin and pregnant animals. Furthermore, using Dll1 reporter mice, we found that Dll1 expression is enriched in MaSCs, and Dll1+ MaSCs have a greater regenerative potential than Dll1− MaSCs. Lineage tracing with Dll1-Cre-ERT2;dTomato reporter mice revealed that Dll1+ cells can produce both basal and luminal cells. Dll1cKO mice exhibit a significant reduction in the number of mammary gland macrophages. The mammary macrophages have molecular features, including enrichment of Wnt and Notch signaling pathway components, that are distinct from those of macrophages in other tissue. Dll1 binds to Notch2 and Notch3 on mammary macrophages to activate Notch signaling, which is necessary to sustain macrophage numbers in the niche and their MaSC-promoting activity. Using a MaSC-macrophage coculture system, we also showed that MaSC-derived Dll1 induces expression of Wnt ligands—such as Wnt3, Wnt10, and Wnt16—from macrophages, and these ligands feed back to MaSCs to promote their stem cell activities. In vivo experiments involving genetic and pharmacological depletion of macrophages, as well as macrophage-specific deficiency of Notch signaling, further validated the crucial role of mammary macrophages in sustaining MaSC activity.  We identified Notch ligand Dll1 as a marker that is enriched in MaSCs and demonstrated that Dll1+ MaSCs can generate both basal and luminal cells. Our study establishes macrophages as important cellular components of the MaSC niche through intercellular coupling of Notch and Wnt signaling. Dll1 produced from MaSCs activates Notch signaling in macrophages to sustain their numbers and enhance the expression of Wnt ligands, which in turn supports Wnt signaling in MaSCs to maintain stem cell activity. Our study defines a Dll1-mediated MaSC niche in which the survival and function of MaSCs and stromal macrophages are mutually regulated by cross-talk between the two cell types through Notch and Wnt signaling. Dll1+ MaSCs interact with mammary macrophages through Notch and Wnt signaling. Dll1 conditional knockout (Dll1cKO) mice display delayed ductal growth compared with wild-type (WT) mice (compare lower and upper left). In mouse mammary glands (upper right), F4/80+ macrophages (brown) are in close proximity to basal MECs. In the schematic model (lower right), Dll1 in MaSCs (pink) activates Notch signaling in macrophages, increasing their production of Wnt ligands, which in turn promote MaSC activity. The stem cell niche is a specialized environment that dictates stem cell function during development and homeostasis. We show that Dll1, a Notch pathway ligand, is enriched in mammary gland stem cells (MaSCs) and mediates critical interactions with stromal macrophages in the surrounding niche in mouse models. Conditional deletion of Dll1 reduced the number of MaSCs and impaired ductal morphogenesis in the mammary gland. Moreover, MaSC-expressed Dll1 activates Notch signaling in stromal macrophages, increasing their expression of Wnt family ligands such as Wnt3, Wnt10A, and Wnt16, thereby initiating a feedback loop that promotes the function of Dll1-expressing MaSCs. Together, these findings reveal functionally important cross-talk between MaSCs and their macrophageal niche through Dll1-mediated Notch signaling.

title: Phagocytosis and clearance of apoptotic cells is mediated by MER
abstract: Apoptosis is fundamental to the development and maintenance of animal tissues and the immune system. Rapid clearance of apoptotic cells by macrophages is important to inhibit inflammation and autoimmune responses against intracellular antigens. Here we report a new function for Mer, a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family. merkd mice with a cytoplasmic truncation of Mer had macrophages deficient in the clearance of apoptotic thymocytes. This was corrected in chimaeric mice reconstituted with bone marrow from wild-type animals. Primary macrophages isolated from merkd mice showed that the phagocytic deficiency was restricted to apoptotic cells and was independent of Fc receptor-mediated phagocytosis or ingestion of other particles. The inability to clear apoptotic cells adequately may be linked to an increased number of nuclear autoantibodies in merkd mice. Thus, the Mer receptor tyrosine kinase seems to be critical for the engulfment and efficient clearance of apoptotic cells. This has implications for inflammation and autoimmune diseases such as systemic lupus erythematosus.

title: Timing Is Everything: Making Neurons versus Glia in the Developing Cortex
abstract: During development of the mammalian nervous system, neural stem cells generate neurons first and glia second, thereby allowing the initial establishment of neural circuitry, and subsequent matching of glial numbers and position to that circuitry. Here, we have reviewed work addressing the mechanisms underlying this timed cell genesis, with a particular focus on the developing cortex. These studies have defined an intriguing interplay between intrinsic epigenetic status, transcription factors, and environmental cues, all of which work together to establish this fascinating and complex biological timing mechanism.

title: Impact of Cytokines on Neural Stem/Progenitor Cell Fate
abstract: Neural stem/progenitor cells (NSC/NPC) can be a powerful tool for the neural repair of the damaged brain. Many of the current challenges with stem cell therapies revolve around the problem that stem cells do not survive, migrate, proliferate and differentiate as much as hoped. Understanding the interaction between NPCs and the immune system is essential for the effective use of stem cell transplantation. Cytokines play an important role in determining the inflammatory microenvironment and have also been shown to have effects on the differentiation, proliferation, migration and survival of NPCs. The effects of cytokines on neural stem cell fate is more complex than once believed; the distinction between anti-inflammatory and pro- inflammatory cytokines is not straightforward and varies based on conditions such as cytokine concentration and area of transplantation. If their role is understood, cytokines could be used to improve the efficacy of stem cell treatments and enhance neural repair. In this review, we provide a comprehensive overview of the effects of various cytokines on NPC fate. The ultimate goal of this review is to demonstrate how manipulation of the CNS microenvironment through alteration of various cytokines can enhance the capacity of NPC differentiation, proliferation, and overall neural repair.

title: Metabolic Induction of Trained Immunity through the Mevalonate Pathway
abstract: Innate immune cells can develop long-term memory after stimulation by microbial products during infections or vaccinations. Here, we report that metabolic signals can induce trained immunity. Pharmacological and genetic experiments reveal that activation of the cholesterol synthesis pathway, but not the synthesis of cholesterol itself, is essential for training of myeloid cells. Rather, the metabolite mevalonate is the mediator of training via activation of IGF1-R and mTOR and subsequent histone modifications in inflammatory pathways. Statins, which block mevalonate generation, prevent trained immunity induction. Furthermore, monocytes of patients with hyper immunoglobulin D syndrome (HIDS), who are mevalonate kinase deficient and accumulate mevalonate, have a constitutive trained immunity phenotype at both immunological and epigenetic levels, which could explain the attacks of sterile inflammation that these patients experience. Unraveling the role of mevalonate in trained immunity contributes to our understanding of the pathophysiology of HIDS and identifies novel therapeutic targets for clinical conditions with excessive activation of trained immunity.

title: Microglia Regulate the Number of Neural Precursor Cells in the Developing Cerebral Cortex
abstract: Neurogenesis must be properly regulated to ensure that cell production does not exceed the requirements of the growing cerebral cortex, yet our understanding of mechanisms that restrain neuron production remains incomplete. We investigated the function of microglial cells in the developing cerebral cortex of prenatal and postnatal macaques and rats and show that microglia limit the production of cortical neurons by phagocytosing neural precursor cells. We show that microglia selectively colonize the cortical proliferative zones and phagocytose neural precursor cells as neurogenesis nears completion. We found that deactivating microglia in utero with tetracyclines or eliminating microglia from the fetal cerebral cortex with liposomal clodronate significantly increased the number of neural precursor cells, while activating microglia in utero through maternal immune activation significantly decreased the number of neural precursor cells. These data demonstrate that microglia play a fundamental role in regulating the size of the precursor cell pool in the developing cerebral cortex, expanding our understanding of the mechanisms that regulate cortical development. Furthermore, our data suggest that any factor that alters the number or activation state of microglia in utero can profoundly affect neural development and affect behavioral outcomes.

title: Microglial Phagocytosis of Newborn Cells Is Induced by Endocannabinoids and Sculpts Sex Differences in Juvenile Rat Social Play
abstract: Brain sex differences are established developmentally and generate enduring changes in circuitry and behavior. Steroid-mediated masculinization of the rat amygdala during perinatal development produces higher levels of juvenile rough-and-tumble play by males. This sex difference in social play is highly conserved across mammals, yet the mechanisms by which it is established are unknown. Here, we report that androgen-induced increases in endocannabinoid tone promote microglia phagocytosis during a critical period of amygdala development. Phagocytic microglia engulf more viable newborn cells in males; in females, less phagocytosis allows more astrocytes to survive to the juvenile age. Blocking complement-dependent phagocytosis in males increases astrocyte survival and prevents masculinization of play. Moreover, increased astrocyte density in the juvenile amygdala reduces neuronal excitation during play. These findings highlight novel mechanisms of brain development whereby endocannabinoids induce microglia phagocytosis to regulate newborn astrocyte number and shape the sexual differentiation of social circuitry and behavior.

title: Two to Tango: Dialog between Immunity and Stem Cells in Health and Disease
abstract: Stem cells regenerate tissues in homeostasis and under stress. By taking cues from their microenvironment or "niche," they smoothly transition between these states. Immune cells have surfaced as prominent members of stem cell niches across the body. Here, we draw parallels between different stem cell niches to explore the context-specific interactions that stem cells have with tissue-resident and recruited immune cells. We also highlight stem cells' innate ability to sense and respond to stress and the enduring memory that forms from such encounters. This fascinating crosstalk holds great promise for novel therapies in inflammatory diseases and regenerative medicine.