Astrogenesis in the murine dentate gyrus is a life-long and dynamic process.

Astrocytes are highly abundant in the mammalian brain, and their functions are of vital importance for all aspects of development, adaption, and aging of the central nervous system (CNS). Mounting evidence indicates the important contributions of astrocytes to a wide range of neuropathies. Still, our understanding of astrocyte development significantly lags behind that of other CNS cells. We here combine immunohistochemical approaches with genetic fate-mapping, behavioural paradigms, single-cell transcriptomics, and in vivo two-photon imaging, to comprehensively assess the generation and the proliferation of astrocytes in the dentate gyrus (DG) across the life span of a mouse. Astrogenesis in the DG is initiated by radial glia-like neural stem cells giving rise to locally dividing astrocytes that enlarge the astrocyte compartment in an outside-in-pattern. Also in the adult DG, the vast majority of astrogenesis is mediated through the proliferation of local astrocytes. Interestingly, locally dividing astrocytes were able to adapt their proliferation to environmental and behavioral stimuli revealing an unexpected plasticity. Our study establishes astrocytes as enduring plastic elements in DG circuits, implicating a vital contribution of astrocyte dynamics to hippocampal plasticity.

Astrocytes in the adult dentate gyrus-balance between adult and developmental tasks.

Astrocytes, a major glial cell type in the brain, are indispensable for the integration, maintenance and survival of neurons during development and adulthood. Both life phases make specific demands on the molecular and physiological properties of astrocytes, and most research projects traditionally focus on either developmental or adult astrocyte functions. In most brain regions, the generation of brain cells and the establishment of neural circuits ends with postnatal development. However, few neurogenic niches exist in the adult brain in which new neurons and glial cells are produced lifelong, and the integration of new cells into functional circuits represent a very special form of plasticity. Consequently, in the neurogenic niche, the astrocytes must be equipped to execute both mature and developmental tasks in order to integrate newborn neurons into the circuit and yet maintain overall homeostasis without affecting the preexisting neurons. In this review, we focus on astrocytes of the hippocampal dentate gyrus (DG), and discuss specific features of the astrocytic compartment that may allow the execution of both tasks. Firstly, astrocytes of the adult DG are molecularly, morphologically and functionally diverse, and the distinct astrocytes subtypes are characterized by their localization to DG layers. This spatial separation may lead to a functional specification of astrocytes subtypes according to the neuronal structures they are embedded in, hence a division of labor. Secondly, the astrocytic compartment is not static, but steadily increasing in numbers due to lifelong astrogenesis. Interestingly, astrogenesis can adapt to environmental and behavioral stimuli, revealing an unexpected astrocyte dynamic that allows the niche to adopt to changing demands. The diversity and dynamic of astrocytes in the adult DG implicate a vital contribution to hippocampal plasticity and represent an interesting model to uncover mechanisms how astrocytes simultaneously fulfill developmental and adult tasks.

Four-and-a-Half LIM-Domain Protein 2 (FHL2) Induces Neuropeptide Y (NPY) in Macrophages in Visceral Adipose Tissue and Promotes Diet-Induced Obesity.

Obesity is characterized by the expansion of the adipose tissue, usually accompanied by inflammation, with a prominent role of macrophages infiltrating the visceral adipose tissue (VAT). This chronic inflammation is a major driver of obesity-associated comorbidities. Four-and-a-half LIM-domain protein 2 (FHL2) is a multifunctional adaptor protein that is involved in the regulation of various biological functions and the maintenance of the homeostasis of different tissues. In this study, we aimed to gain new insights into the expression and functional role of FHL2 in VAT in diet-induced obesity. We found enhanced FHL2 expression in the VAT of mice with Western-type diet (WTD)-induced obesity and obese humans and identified macrophages as the cellular source of enhanced FHL2 expression in VAT. In mice with FHL2 deficiency (FHL2KO), WTD feeding resulted in reduced body weight gain paralleled by enhanced energy expenditure and uncoupling protein 1 (UCP1) expression, indicative of activated thermogenesis. In human VAT, FHL2 was inversely correlated with UCP1 expression. Furthermore, macrophage infiltration and the expression of the chemokine MCP-1, a known promotor of macrophage accumulation, was significantly reduced in WTD-fed FHL2KO mice compared with wild-type (wt) littermates. While FHL2 depletion did not affect the differentiation or lipid metabolism of adipocytes in vitro, FHL2 depletion in macrophages resulted in reduced expressions of MCP-1 and the neuropeptide Y (NPY). Furthermore, WTD-fed FHL2KO mice showed reduced NPY expression in VAT compared with wt littermates, and NPY expression was enhanced in VAT resident macrophages of obese individuals. Stimulation with recombinant NPY induced not only UCP1 expression and lipid accumulation but also MCP-1 expression in adipocytes. Collectively, these findings indicate that FHL2 is a positive regulator of NPY and MCP-1 expression in macrophages and herewith closely linked to the mechanism of obesity-associated lipid accumulation and inflammation in VAT. Thus, FHL2 appears as a potential novel target to interfere with the macrophage-adipocyte crosstalk in VAT for treating obesity and related metabolic disorders.

Heterogeneity of Radial Glia-Like Cells in the Adult Hippocampus.

Adult neurogenesis is tightly regulated by the neurogenic niche. Cellular contacts between niche cells and neural stem cells are hypothesized to regulate stem cell proliferation or lineage choice. However, the structure of adult neural stem cells and the contact they form with niche cells are poorly described. Here, we characterized the morphology of radial glia-like (RGL) cells, their molecular identity, proliferative activity, and fate determination in the adult mouse hippocampus. We found the coexistence of two morphotypes of cells with prototypical morphological characteristics of RGL stem cells: Type α cells, which represented 76% of all RGL cells, displayed a long primary process modestly branching into the molecular layer and type ß cells, which represented 24% of all RGL cells, with a shorter radial process highly branching into the outer granule cell layer-inner molecular layer border. Stem cell markers were expressed in type α cells and coexpressed with astrocytic markers in type ß cells. Consistently, in vivo lineage tracing indicated that type α cells can give rise to neurons, astrocytes, and type ß cells, whereas type ß cells do not proliferate. Our results reveal that the adult subgranular zone of the dentate gyrus harbors two functionally different RGL cells, which can be distinguished by simple morphological criteria, supporting a morphofunctional role of their thin cellular processes. Type ß cells may represent an intermediate state in the transformation of type α, RGL stem cells, into astrocytes.

Dickkopf 3 Promotes the Differentiation of a Rostrolateral Midbrain Dopaminergic Neuronal Subset In Vivo and from Pluripotent Stem Cells In Vitro in the Mouse.

Wingless-related MMTV integration site 1 (WNT1)/ß-catenin signaling plays a crucial role in the generation of mesodiencephalic dopaminergic (mdDA) neurons, including the substantia nigra pars compacta (SNc) subpopulation that preferentially degenerates in Parkinson's disease (PD). However, the precise functions of WNT1/ß-catenin signaling in this context remain unknown. Stem cell-based regenerative (transplantation) therapies for PD have not been implemented widely in the clinical context, among other reasons because of the heterogeneity and incomplete differentiation of the transplanted cells. This might result in tumor formation and poor integration of the transplanted cells into the dopaminergic circuitry of the brain. Dickkopf 3 (DKK3) is a secreted glycoprotein implicated in the modulation of WNT/ß-catenin signaling. Using mutant mice, primary ventral midbrain cells, and pluripotent stem cells, we show that DKK3 is necessary and sufficient for the correct differentiation of a rostrolateral mdDA neuron subset. Dkk3 transcription in the murine ventral midbrain coincides with the onset of mdDA neurogenesis and is required for the activation and/or maintenance of LMX1A (LIM homeobox transcription factor 1α) and PITX3 (paired-like homeodomain transcription factor 3) expression in the corresponding mdDA precursor subset, without affecting the proliferation or specification of their progenitors. Notably, the treatment of differentiating pluripotent stem cells with recombinant DKK3 and WNT1 proteins also increases the proportion of mdDA neurons with molecular SNc DA cell characteristics in these cultures. The specific effects of DKK3 on the differentiation of rostrolateral mdDA neurons in the murine ventral midbrain, together with its known prosurvival and anti-tumorigenic properties, make it a good candidate for the improvement of regenerative and neuroprotective strategies in the treatment of PD. Significance statement: We show here that Dickkopf 3 (DKK3), a secreted modulator of WNT (Wingless-related MMTV integration site)/ß-catenin signaling, is both necessary and sufficient for the proper differentiation and survival of a rostrolateral (parabrachial pigmented nucleus and dorsomedial substantia nigra pars compacta) mesodiencephalic dopaminergic neuron subset, using Dkk3 mutant mice and murine primary ventral midbrain and pluripotent stem cells. The progressive loss of these dopamine-producing mesodiencephalic neurons is a hallmark of human Parkinson's disease, which can up to now not be halted by clinical treatments of this disease. Thus, the soluble DKK3 protein might be a promising new agent for the improvement of current protocols for the directed differentiation of pluripotent and multipotent stem cells into mesodiencephalic dopaminergic neurons and for the promotion of their survival in situ.

Continuous live imaging of adult neural stem cell division and lineage progression in vitro.

Little is known about the intrinsic specification of adult neural stem cells (NSCs) and to what extent they depend on their local niche. To observe adult NSC division and lineage progression independent of their niche, we isolated cells from the adult mouse subependymal zone (SEZ) and cultured them at low density without growth factors. We demonstrate here that SEZ cells in this culture system are primarily neurogenic and that adult NSCs progress through stereotypic lineage trees consisting of asymmetric stem cell divisions, symmetric transit-amplifying divisions and final symmetric neurogenic divisions. Stem cells, identified by their astro/radial glial identity and their slow-dividing nature, were observed to generate asymmetrically and fast-dividing cells that maintained an astro/radial glia identity. These, in turn, gave rise to symmetrically and fast-dividing cells that lost glial hallmarks, but had not yet acquired neuronal features. The number of amplifying divisions was limited to a maximum of five in this system. Moreover, we found that cell growth correlated with the number of subsequent divisions of SEZ cells, with slow-dividing astro/radial glia exhibiting the most substantial growth prior to division. The fact that in the absence both of exogenously supplied growth factors and of signals provided by the local niche neurogenic lineage progression takes place in such stereotypic fashion, suggests that lineage progression is, to a significant degree, cell intrinsic or pre-programmed at the beginning of the lineage.

Sox9 regulates melanocytic fate decision of adult hair follicle stem cells.

The bulge of hair follicles harbors Nestin+ (neural crest like) stem cells, which exhibit the potential to generate various cell types including melanocytes. In this study, we aimed to determine the role of Sox9, an important regulator during neural crest development, in melanocytic differentiation of those adult Nestin+ cells. Immunohistochemical analysis after conditional Sox9 deletion in Nestin+ cells of adult mice revealed that Sox9 is crucial for melanocytic differentiation of these cells and that Sox9 acts as a fate determinant between melanocytic and glial fate. A deeper understanding of factors that regulate fate decision, proliferation and differentiation of these stem cells provides new aspects to melanoma research as melanoma cells share many similarities with neural crest cells. In summary, we here show the important role of Sox9 in melanocytic versus glial fate decision of Nestin+ stem cells in the skin of adult mice.

NR2F1 shapes mitochondria in the mouse brain, providing new insights into Bosch-Boonstra-Schaaf optic atrophy syndrome.

The nuclear receptor NR2F1 acts as a strong transcriptional regulator in embryonic and postnatal neural cells. In humans, mutations in the NR2F1 gene cause Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS), a rare neurodevelopmental disorder characterized by multiple clinical features including vision impairment, intellectual disability and autistic traits. In this study, we identified, by genome-wide and in silico analyses, a set of nuclear-encoded mitochondrial genes as potential genomic targets under direct NR2F1 transcriptional control in neurons. By combining mouse genetic, neuroanatomical and imaging approaches, we demonstrated that conditional NR2F1 loss of function within the adult mouse hippocampal neurogenic niche results in a reduced mitochondrial mass associated with mitochondrial fragmentation and downregulation of key mitochondrial proteins in newborn neurons, the genesis, survival and functional integration of which are impaired. Importantly, we also found dysregulation of several nuclear-encoded mitochondrial genes and downregulation of key mitochondrial proteins in the brain of Nr2f1-heterozygous mice, a validated BBSOAS model. Our data point to an active role for NR2F1 in the mitochondrial gene expression regulatory network in neurons and support the involvement of mitochondrial dysfunction in BBSOAS pathogenesis.

Two novel CreERT2 transgenic mouse lines to study melanocytic cells in vivo.

The skin of adult mammals protects from radiation, physical and chemical insults. While melanocytes and melanocyte-producing stem cells contribute to proper skin function in healthy organisms, dysfunction of these cells can lead to the generation of malignant melanoma-the deadliest type of skin cancer. Addressing cells of the melanocyte lineage in vivo represents a prerequisite for the understanding of melanoma on cellular level and the development of preventive and treatment strategies. Here, the inducible Cre-loxP-system has emerged as a promising tool to specifically target, monitor, and modulate cells in adult mice. Re-analysis of existing sequencing data sets of melanocytic cells revealed that genes with a known function in neural cells, including neural stem cells (Aldh1L1 and Nestin), are also expressed in melanocytic cells. Therefore, in this study, we explored whether the promoter activity of Nestin and Aldh1L1 can serve to target cells of the melanocyte lineage using the inducible CreERT2 -loxP-system. Using an immunohistochemical approach and different time points of analysis, we were able to map the melanocytic fate of recombined stem cells in the adult hair follicle of Nestin-CreERT2 and Aldh1L1-CreERT2 transgenic mice. Thus, we here present two new mouse models and propose their use to study and putatively modulate adult melanocytic cells in vivo.

Dentate gyrus astrocytes exhibit layer-specific molecular, morphological and physiological features.

Neuronal heterogeneity has been established as a pillar of higher central nervous system function, but glial heterogeneity and its implications for neural circuit function are poorly understood. Here we show that the adult mouse dentate gyrus (DG) of the hippocampus is populated by molecularly distinct astrocyte subtypes that are associated with distinct DG layers. Astrocytes localized to different DG compartments also exhibit subtype-specific morphologies. Physiologically, astrocytes in upper DG layers form large syncytia, while those in lower DG compartments form smaller networks. Astrocyte subtypes differentially express glutamate transporters, which is associated with different amplitudes of glutamate transporter-mediated currents. Key molecular and morphological features of astrocyte diversity in the mice DG are conserved in humans. This adds another layer of complexity to our understanding of brain network composition and function, which will be crucial for further studies on astrocytes in health and disease.

Distribution of Aldh1L1-CreERT2 Recombination in Astrocytes Versus Neural Stem Cells in the Neurogenic Niches of the Adult Mouse Brain.

In the adult central nervous system, neural stem cells (NSCs) reside in two discrete niches: the subependymal zone (SEZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG). Here, NSCs represent a population of highly specialized astrocytes that are able to proliferate and give rise to neuronal and glial progeny. This process, termed adult neurogenesis, is extrinsically regulated by other niche cells such as non-stem cell astrocytes. Studying these non-stem cell niche astrocytes and their role during adult neuro- and gliogenesis has been hampered by the lack of genetic tools to discriminate between transcriptionally similar NSCs and niche astrocytes. Recently, Aldh1L1 has been shown to be a pan-astrocyte marker and that its promoter can be used to specifically target astrocytes using the Cre-loxP system. In this study we explored whether the recently described Aldh1L1-CreERT2 mouse line (Winchenbach et al., 2016) can serve to specifically target niche astrocytes without inducing recombination in NSCs in adult neurogenic niches. Using short- and long-term tamoxifen protocols we revealed high recombination efficiency and specificity in non-stem cell astrocytes and little to no recombination in NSCs of the adult DG. However, in the SEZ we observed recombination in ependymal cells, astrocytes, and NSCs, the latter giving rise to neuronal progeny of the rostral migratory stream and olfactory bulb. Thus, we recommend the here described Aldh1L1-CreERT2 mouse line for predominantly studying the functions of non-stem cell astrocytes in the DG under physiological and pathological conditions.

Untangling human neurogenesis to understand and counteract brain disorders.

Neurogenesis in the human postnatal brain occurs in two regions, the subventricular zone of the later ventricle and the dentate gyrus of the hippocampus. While it is well accepted that SVZ and hippocampal neurogenesis are active during juvenile stages in human, their contribution during adulthood and ageing as well as pathological states is recently animating the neural stem cell research field. In this review we will discuss recent evidence about the organization of SVZ and hippocampal neurogenic niches, and will report on how human adult neurogenesis may contribute to disease and appears to respond to neurodegeneration. In light of these novel findings, we will discuss how we can target human adult neurogenesis in order to influence brain disease trajectories.

Role of Astrocytes in the Neurogenic Niches.

In the mammalian brain, highly specialized astrocytes serve as neural stem cells (NSCs) that divide and give rise to new neurons, in a process called neurogenesis. During embryonic development NSCs generate almost all neurons of the brain. Soon after birth the neurogenic potential of NSCs is highly reduced, and neurogenesis occurs only in two specialized brain regions called the neurogenic niches. Niche cells are essential to stem cells as they provide structural and nutritional support, and control fundamental stem cell decisions. Astrocytes, major components of the adult neurogenic niches, are evolving as important regulators of neurogenesis, by controlling NSC proliferation, fate choice, and differentiation of the progeny. Therefore, astrocytes contribute to neurogenesis in two ways: as NSCs and as niche cells. This review highlights the role of astrocyte-like NSCs during development and adulthood, and summarizes how niche astrocytes control the process of adult neurogenesis.

Mitochondrial Dysfunction in Astrocytes Impairs the Generation of Reactive Astrocytes and Enhances Neuronal Cell Death in the Cortex Upon Photothrombotic Lesion.

Mitochondria are key organelles in regulating the metabolic state of a cell. In the brain, mitochondrial oxidative metabolism is the prevailing mechanism for neurons to generate ATP. While it is firmly established that neuronal function is highly dependent on mitochondrial metabolism, it is less well-understood how astrocytes function rely on mitochondria. In this study, we investigate if astrocytes require a functional mitochondrial electron transport chain (ETC) and oxidative phosphorylation (oxPhos) under physiological and injury conditions. By immunohistochemistry we show that astrocytes expressed components of the ETC and oxPhos complexes in vivo. Genetic inhibition of mitochondrial transcription by conditional deletion of mitochondrial transcription factor A (Tfam) led to dysfunctional ETC and oxPhos activity, as indicated by aberrant mitochondrial swelling in astrocytes. Mitochondrial dysfunction did not impair survival of astrocytes, but caused a reactive gliosis in the cortex under physiological conditions. Photochemically initiated thrombosis induced ischemic stroke led to formation of hyperfused mitochondrial networks in reactive astrocytes of the perilesional area. Importantly, mitochondrial dysfunction significantly reduced the generation of new astrocytes and increased neuronal cell death in the perilesional area. These results indicate that astrocytes require a functional ETC and oxPhos machinery for proliferation and neuroprotection under injury conditions.

FoxO Function Is Essential for Maintenance of Autophagic Flux and Neuronal Morphogenesis in Adult Neurogenesis.

Autophagy is a conserved catabolic pathway with emerging functions in mammalian neurodevelopment and human neurodevelopmental diseases. The mechanisms controlling autophagy in neuronal development are not fully understood. Here, we found that conditional deletion of the Forkhead Box O transcription factors FoxO1, FoxO3, and FoxO4 strongly impaired autophagic flux in developing neurons of the adult mouse hippocampus. Moreover, FoxO deficiency led to altered dendritic morphology, increased spine density, and aberrant spine positioning in adult-generated neurons. Strikingly, pharmacological induction of autophagy was sufficient to correct abnormal dendrite and spine development of FoxO-deficient neurons. Collectively, these findings reveal a novel link between FoxO transcription factors, autophagic flux, and maturation of developing neurons.

Newborn Neurons in the Aged Hippocampus-Scarce and Slow but Highly Plastic.

In this issue of Cell Reports, Trinchero et al. (2017) demonstrate that newborn neurons in the aged hippocampus are delayed in development but are highly susceptible to stimuli improving neuronal activity. This plasticity is mediated cell-intrinsically by neurotrophin signaling.

Mitochondrial Metabolism-Mediated Regulation of Adult Neurogenesis.

The life-long generation of new neurons from radial glia-like neural stem cells (NSCs) is achieved through a stereotypic developmental sequence that requires precise regulatory mechanisms to prevent exhaustion or uncontrolled growth of the stem cell pool. Cellular metabolism is the new kid on the block of adult neurogenesis research and the identity of stage-specific metabolic programs and their impact on neurogenesis turns out to be an emerging research topic in the field. Mitochondrial metabolism is best known for energy production but it contains a great deal more. Mitochondria are key players in a variety of cellular processes including ATP synthesis through functional coupling of the electron transport chain and oxidative phosphorylation, recycling of hydrogen carriers, biosynthesis of cellular building blocks, and generation of reactive oxygen species that can modulate signaling pathways in a redox-dependent fashion. In this review, I will discuss recent findings describing stage-specific modulations of mitochondrial metabolism within the adult NSC lineage, emphasizing its importance for NSC self-renewal, proliferation of neural stem and progenitor cells (NSPCs), cell fate decisions, and differentiation and maturation of newborn neurons. I will furthermore summarize the important role of mitochondrial dysfunction in tissue regeneration and ageing, suggesting it as a potential therapeutic target for regenerative medicine practice.

Synergic Functions of miRNAs Determine Neuronal Fate of Adult Neural Stem Cells.

Adult neurogenesis requires the precise control of neuronal versus astrocyte lineage determination in neural stem cells. While microRNAs (miRNAs) are critically involved in this step during development, their actions in adult hippocampal neural stem cells (aNSCs) has been unclear. As entry point to address that question we chose DICER, an endoribonuclease essential for miRNA biogenesis and other RNAi-related processes. By specific ablation of Dicer in aNSCs in vivo and in vitro, we demonstrate that miRNAs are required for the generation of new neurons, but not astrocytes, in the adult murine hippocampus. Moreover, we identify 11 miRNAs, of which 9 have not been previously characterized in neurogenesis, that determine neurogenic lineage fate choice of aNSCs at the expense of astrogliogenesis. Finally, we propose that the 11 miRNAs sustain adult hippocampal neurogenesis through synergistic modulation of 26 putative targets from different pathways.

Role of Mitochondrial Metabolism in the Control of Early Lineage Progression and Aging Phenotypes in Adult Hippocampal Neurogenesis.

Precise regulation of cellular metabolism is hypothesized to constitute a vital component of the developmental sequence underlying the life-long generation of hippocampal neurons from quiescent neural stem cells (NSCs). The identity of stage-specific metabolic programs and their impact on adult neurogenesis are largely unknown. We show that the adult hippocampal neurogenic lineage is critically dependent on the mitochondrial electron transport chain and oxidative phosphorylation machinery at the stage of the fast proliferating intermediate progenitor cell. Perturbation of mitochondrial complex function by ablation of the mitochondrial transcription factor A (Tfam) reproduces multiple hallmarks of aging in hippocampal neurogenesis, whereas pharmacological enhancement of mitochondrial function ameliorates age-associated neurogenesis defects. Together with the finding of age-associated alterations in mitochondrial function and morphology in NSCs, these data link mitochondrial complex function to efficient lineage progression of adult NSCs and identify mitochondrial function as a potential target to ameliorate neurogenesis-defects in the aging hippocampus.

Transcription-Factor-Dependent Control of Adult Hippocampal Neurogenesis.

Adult-generated dentate granule neurons have emerged as major contributors to hippocampal plasticity. New neurons are generated from neural stem cells through a complex sequence of proliferation, differentiation, and maturation steps. Development of the new neuron is dependent on the precise temporal activity of transcription factors, which coordinate the expression of stage-specific genetic programs. Here, we review current knowledge in transcription factor-mediated regulation of mammalian neural stem cells and neurogenesis and will discuss potential mechanisms of how transcription factor networks, on one hand, allow for precise execution of the developmental sequence and, on the other hand, allow for adaptation of the rate and timing of adult neurogenesis in response to complex stimuli. Understanding transcription factor-mediated control of neuronal development will provide new insights into the mechanisms underlying neurogenesis-dependent plasticity in health and disease.