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Background RNA-binding proteins (RBPs) function as master regulators of gene expression. Alterations in RBP expression and function are often observed in cancer and influence critical pathways implicated in tumor initiation and growth. Identification and characterization of oncogenic RBPs and their regulatory networks provide new opportunities for targeted therapy. Results We identify the RNA-binding protein SERBP1 as a novel regulator of glioblastoma (GBM) development. High SERBP1 expression is prevalent in GBMs and correlates with poor patient survival and poor response to chemo- and radiotherapy. SERBP1 knockdown causes delay in tumor growth and impacts cancer-relevant phenotypes in GBM and glioma stem cell lines. RNAcompete identifies a GC-rich region as SERBP1-binding motif; subsequent genomic and functional analyses establish SERBP1 regulation role in metabolic routes preferentially used by cancer cells. An important consequence of these functions is SERBP1 impact on methionine production. SERBP1 knockdown decreases methionine levels causing a subsequent reduction in histone methylation as shown for H3K27me3 and upregulation of genes associated with neurogenesis, neuronal differentiation, and function. Further analysis demonstrates that several of these genes are downregulated in GBM, potentially through epigenetic silencing as indicated by the presence of H3K27me3 sites. Conclusions SERBP1 is the first example of an RNA-binding protein functioning as a central regulator of cancer metabolism and indirect modulator of epigenetic regulation in GBM. By bridging these two processes, SERBP1 enhances glioma stem cell phenotypes and contributes to GBM poorly differentiated state.

Microglia have been implicated in multiple sclerosis (MS) pathogenesis. The fractalkine receptor CX3CR1 regulates the activation of pathogenic microglia in models of MS and the human polymorphic CX3CR1I249/M280 (hCX3CR1I249/M280) variant increases MS disease progression. However, the role of hCX3CR1I249/M280 on microglial activation and central nervous system repair and regenerative mechanisms remain unknown. Therefore, using transgenic mice expressing the hCX3CR1I249/M280 variant, we aimed to determine the contribution of defective CX3CR1 signaling to remyelination and neurogenesis in the cuprizone model of focal demyelination. Here, we report that mice expressing hCX3CR1I249/M280 exhibit marked demyelination and microgliosis follow acute cuprizone treatment. Cuprizone-treated CX3CR1-deficient and fractalkine-deficient mice displayed a comparable phenotype. Nanostring gene expression analysis in demyelinated lesions showed that hCX3CR1I249/M280 upregulates genes associated with inflammation, oxidative stress and disease-associated microglia. In addition, gene expression analysis in the subgranular zone (SGZ) of the hippocampus in hCX3CR1I249/M280 mice was associated with a significant downregulation of gene networks linked to neurogenesis following acute demyelination. Confocal microscopy showed that hCX3CR1I249/M280 or loss of CX3CR1 signaling inhibits the generation of progeny from the neurogenic niche, including cells involved in myelin repair. These results provide evidence for the pathogenic capacity of hCX3CR1I249/M280 on microglia dysfunction and therapeutic targeting of CX3CR1 to promote CNS repair in MS.

RNA-binding proteins (RBPs) and miRNAs are critical gene expression regulators that interact with one another in cooperative and antagonistic fashions. We identified Musashi1 (Msi1) and miR-137 as regulators of a molecular switch between self-renewal and differentiation. Msi1 and miR-137 have opposite expression patterns and functions, and Msi1 is repressed by miR-137. Msi1 is a stem–cell protein implicated in self-renewal while miR-137 functions as a proneuronal differentiation miRNA. In gliomas, miR-137 functions as a tumor suppressor while Msi1 is a prooncogenic factor. We suggest that the balance between Msi1 and miR-137 is a key determinant in cell fate decisions and disruption of this balance could contribute to neurodegenerative diseases and glioma development. Genomic analyses revealed that Msi1 and miR-137 share 141 target genes associated with differentiation, development, and morphogenesis. Initial results pointed out that these two regulators have an opposite impact on the expression of their target genes. Therefore, we propose an antagonistic model in which this network of shared targets could be either repressed by miR-137 or activated by Msi1, leading to different outcomes (self-renewal, proliferation, tumorigenesis).

The brain can generate new neurons from neural stem cells throughout life. However, the capacity for neurogenesis declines with age, reducing the potential for learning and repair. We explored the effects of calorie restriction, an established anti-aging intervention, on neural stem cells in the subventricular zone of young and aged mice. Calorie restriction transiently enhanced proliferation of neural progenitor cells in young, but not aged mice. However, calorie restriction prevented the age-related loss of neurogenesis in the aged brain. Calorie-restricted mice showed enhanced olfactory memory compared with ad libitum-fed controls, suggesting that calorie restriction can produce functional improvements in the aged brain. Calorie restriction also mitigated the age-related activation of microglia and subsequent increase in pro-inflammatory cytokines. Likewise, calorie restriction prevented increases in senescent cells normally observed in the subventricular zone in aged mice, further protecting this neurogenic niche from pro-inflammatory signals. Together, these data suggest that calorie restriction protects the subventricular zone microenvironment from age-related inflammation, thereby preserving neurogenesis into old age.

Simple Summary miRNAs function as critical regulators of gene expression and have been defined as contributors of cancer phenotypes by acting as oncogenes or tumor suppressors. Based on these findings, miRNA-based therapies have been explored in the treatment of many different malignancies. The use of single miRNAs has faced some challenges and showed limited success. miRNAs cooperate to regulate distinct biological processes and pathways and, therefore, combination of related miRNAs could amplify the repression of oncogenic factors and the effect on cancer relevant pathways. We established that the combination of tumor suppressor miRNAs miR-124, miR-128, and miR-137 is much more effective than single miRNAs in disrupting proliferation and survival of glioma stem cells and neuroblastoma lines and promoting differentiation and response to radiation. Subsequent genomic analyses showed that other combinations of tumor suppressor miRNAs could be equally effective, and its use could provide new routes to target in special cancer-initiating cell populations. Abstract Tumor suppressor microRNAs (miRNAs) have been explored as agents to target cancer stem cells. Most strategies use a single miRNA mimic and present many disadvantages, such as the amount of reagent required and the diluted effect on target genes. miRNAs work in a cooperative fashion to regulate distinct biological processes and pathways. Therefore, we propose that miRNA combinations could provide more efficient ways to target cancer stem cells. We have previously shown that miR-124, miR-128, and miR-137 function synergistically to regulate neurogenesis. We used a combination of these three miRNAs to treat glioma stem cells and showed that this treatment was much more effective than single miRNAs in disrupting cell proliferation and survival and promoting differentiation and response to radiation. Transcriptomic analyses indicated that transcription regulation, angiogenesis, metabolism, and neuronal differentiation are among the main biological processes affected by transfection of this miRNA combination. In conclusion, we demonstrated the value of using combinations of neurogenic miRNAs to disrupt cancer phenotypes and glioma stem cell growth. The synergistic effect of these three miRNA amplified the repression of oncogenic factors and the effect on cancer relevant pathways. Future therapeutic approaches would benefit from utilizing miRNA combinations, especially when targeting cancer-initiating cell populations.

Adult neurogenesis is the process of producing new neurons from neural stem cells (NSCs) for integration into the brain circuitry. Neurogenesis occurs throughout life in the ventricular-subventricular zone (V-SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampal dentate gyrus. However, during aging, NSCs and their progenitors exhibit reduced proliferation and neuron production, which is thought to contribute to age-related cognitive impairment and reduced plasticity that is necessary for some types of brain repair. In this review, we describe NSCs and their niches during tissue homeostasis and how they undergo age-associated remodeling and dysfunction. We also discuss some of the functional ramifications in the brain from NSC aging. Finally, we discuss some recent insights from interventions in NSC aging that could eventually translate into therapies for healthy brain aging.

Aging is a negative regulator of general homeostasis, tissue function, and regeneration. Changes in organismal energy levels and physiology, through systemic manipulations such as calorie restriction and young blood infusion, can regenerate tissue activity and increase lifespan in aged mice. However, whether these two systemic manipulations could be linked has never been investigated. Here, we report that systemic GDF11 triggers a calorie restriction‐like phenotype without affecting appetite or GDF15 levels in the blood, restores the insulin/IGF‐1 signaling pathway, and stimulates adiponectin secretion from white adipose tissue by direct action on adipocytes, while repairing neurogenesis in the aged brain. These findings suggest that GDF11 has a pleiotropic effect on an organismal level and that it could be a linking mechanism of rejuvenation between heterochronic parabiosis and calorie restriction. As such, GDF11 could be considered as an important therapeutic candidate for age‐related neurodegenerative and metabolic disorders., GDF11 stimulates adipocytes to release adiponectin in the bloodstream of aged mice and induces a calorie restriction‐like phenotype without affecting appetite. At the same time, these same GDF11‐treated aged mice exhibit enhanced neurogenesis. This shows that GDF11 has pleiotropic effects in both the brain and the periphery of aged mice, linking the beneficial effects of both heterochronic parabiosis and calorie restriction.

13-Cis-retinoic acid (RA) is typically used in postremission maintenance therapy in patients with neuroblastoma. However, side effects and recurrence are often observed. We investigated the use of miRNAs as a strategy to replace RA as promoters of differentiation. miR-124 was identified as the top candidate in a functional screen. Genomic target analysis indicated that repression of a network of transcription factors (TF) could be mediating most of miR-124's effect in driving differentiation. To advance miR-124 mimic use in therapy and better define its mechanism of action, a high-throughput siRNA morphologic screen focusing on its TF targets was conducted and ELF4 was identified as a leading candidate for miR-124 repression. By altering its expression levels, we showed that ELF4 maintains neuroblastoma in an undifferentiated state and promotes proliferation. Moreover, ELF4 transgenic expression was able to counteract the neurogenic effect of miR-124 in neuroblastoma cells. With RNA sequencing, we established the main role of ELF4 to be regulation of cell-cycle progression, specifically through the DREAM complex. Interestingly, several cell-cycle genes activated by ELF4 are repressed by miR-124, suggesting that they might form a TF–miRNA regulatory loop. Finally, we showed that high ELF4 expression is often observed in neuroblastomas and is associated with poor survival. Implications: miR-124 induces neuroblastoma differentiation partially through the downregulation of TF ELF4, which drives neuroblastoma proliferation and its undifferentiated phenotype.

Neural stem cells exist in specialized regions of the brain and have the capacity to give rise to neurons and glia over the lifespan. The process of giving rise to new neurons, also known as neurogenesis, is thought to be important in cognition and certain types of brain repair. However, during aging, neural stem cell number and function is reduced resulting in fewer new neurons and declines in learning, memory and repair. Recently, research has approached this problem through the lens of rejuvenation that now has produced several strategies, from dietary to pharmacological interventions, to restore functional neurogenesis that resembles the youthful brain. Here, we outline aging in the subventricular zone neurogenic niche, review the multiple modalities of rejuvenation strategies, and propose next steps for future studies to approach translational outcomes.

Neural stem cells (NSCs) exist throughout life in the ventricular–subventricular zone (V-SVZ) of the mammalian forebrain. During aging NSC function is diminished through an unclear mechanism. In this study, we establish microglia, the immune cells of the brain, as integral niche cells within the V-SVZ that undergo age-associated repositioning in the V-SVZ. Microglia become activated early before NSC deficits during aging resulting in an antineurogenic microenvironment due to increased inflammatory cytokine secretion. These age-associated changes were not observed in non-neurogenic brain regions, suggesting V-SVZ microglia are specialized. Using a sustained inflammatory model in young adult mice, we induced microglia activation and inflammation that was accompanied by reduced NSC proliferation in the V-SVZ. Furthermore, in vitro studies revealed secreted factors from activated microglia reduced proliferation and neuron production compared to secreted factors from resting microglia. Our results suggest that age-associated chronic inflammation contributes to declines in NSC function within the aging neurogenic niche.

miRNAs are critical regulators of tumorigenesis, acting as oncogenes or tumor suppressors. Previous work, by us and others, have demonstrated that miR-124, -128, and -137 are key players in controlling neurogenesis and gliomagenesis. Together these miRNAs promote differentiation of neural stem cells (NSCs), while in gliomas their dysregulation contribute to the glioma phenotype. The three miRNAs display strong expression correlation in GBM cohorts and synergize to drive neuronal differentiation of NSCs by regulating a shared network of genes. Individually ectopic overexpression of these miRNAs induces significant changes in proliferation, and differentiation of GBM cells; however, it is unclear if they synergize to produce a stronger effect as observed in NSCs. We tested the combination of miR-124, -128, and -137 in glioblastoma cell lines and found that together they produce a synergistic effect, decreasing cell proliferation and we will test this synergy in other systems. In NSCs we found an interconnected transcription factor (TF) network to be important targets of miR-124, -128, and -137. Due to their importance in NSCs, we hypothesized that these TFs are critical in gliomas. We performed a siRNA screen against the TFs network in GBM cell lines and examined the impact of knockdown on cell proliferation, viability, and apoptosis. While several genes were important in maintaining the glioma phenotype, knockdown of ETS-related transcription factor Elf-4 (ELF4) produced the strongest phenotype changes. We will next characterize ELF4’s transcriptional influence utilizing ChIP-seq paired with RNA-seq. Overall our initial data suggests that this miRNA-Transcription factor network is important in the glioma phenotype, and that combination of the three miRNAs is synergistic. Furthermore, we find the ELF4 is a critical TF from this network and regulates the glioma phenotype.

Neurogenesis in mammals occurs throughout life in two brain regions: the ventricular–subventricular zone (V–SVZ) and the subgranular zone (SGZ) of the hippocampal dentate gyrus. Development and regulation of the V–SVZ and SGZ is unique to each brain region, but with several similar characteristics. Alterations to the production of new neurons in neurogenic regions have been linked to psychiatric and neurodegenerative disorders. Decline in neurogenesis in the SGZ correlates with affective and psychiatric disorders, and can be reversed by antidepressant and antipsychotic drugs. Likewise, neurogenesis in the V–SVZ can also be enhanced by antidepressant drugs. The regulation of neurogenesis by neurotransmitters, particularly monoamines, in both regions suggests that aberrant neurotransmitter signaling observed in psychiatric disease may play a role in the pathology of these mental health disorders. Similarly, the cognitive deficits that accompany neurodegenerative disease may also be exacerbated by decreased neurogenesis. This review explores the regulation and function of neural stem cells in rodents and humans, and the involvement of factors that contribute to psychiatric and cognitive deficits.This article is part of a Special Issue entitled SI:StemsCellsinPsychiatry.

Bone marrow-derived cells (BMDCs) contribute to revascularization after ischemia. However, the mechanisms by which BMDCs support vessel remodeling after cerebral ischemia are not clear. Using mouse chimeras that express enhanced green fluorescent protein in reconstituted bone marrow, we investigated the role of BMDCs in revascularization and brain repair after middle cerebral artery occlusion of murine brain. After ischemia, two populations of BMDCs were observed, one in the brain parenchyma and another associated with the vasculature. The number of BMDCs that infiltrated the brain parenchyma peaked at 7 days and persisted through 14 days, the last time point observed. The majority of BMDCs were characterized as microglia, based on cell-type-specific marker expression. We observed a robust angiogenic response after cerebral ischemia. Bone marrow-derived cells associated with remodeling blood vessels were negative for endothelial markers, but were surrounded by basal lamina and expressed desmin and vimentin, identifying these cells as pericytes. Quantification of BMDCs that expressed desmin revealed increasing desmin expression with time. Perivascular associated BMDCs that expressed desmin were immunoreactive for the angiogenic factors vascular endothelial growth factor and transforming growth factor- β. These findings suggest that pericytes are recruited from the periphery and are involved in blood vessel stabilization during ischemiainduced angiogenesis.

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication in mice results in dopamine neuron degeneration that is alleviated by prevention of microglia cell activation and blockade of iNOS production. However, the role of peripherally derived microglia in this response has not been well characterized. In the present study, we investigated the time course of infiltration and phenotypic differentiation of bone marrow-derived cells (BMDCs) following MPTP treatment in mice, using green fluorescent protein (GFP) bone marrow chimeras. BMDCs were found in the meninges, choroid plexus, blood vessels, and brain parenchyma in both saline and MPTP-treated mice. MPTP stimulated a transient, two-fold increase in the rate of BMDC infiltration into the brain, concomitant with the onset of microglia activation. The majority of BMDCs were microglial in phenotype, as assessed by morphology and expression of the pan-hematopoietic marker CD45 and the microglia marker CD11b. We did not observe BMDCs that expressed neuronal or astroglial markers. Over 90% of bone marrow-derived microglia expressed the inducible form of nitric oxide synthase (iNOS), suggesting that peripherally derived microglia may play a deleterious role in MPTP-induced degeneration.

Neural stem cells (NSCs) persist throughout life in two forebrain areas: the subventricular zone (SVZ) and the hippocampus. Why forebrain NSCs self-renew more extensively than those from other regions remains unclear. Prior studies have shown that the polycomb factor Bmi-1 is necessary for NSC self-renewal and that it represses the cell cycle inhibitors p16, p19, and p21. Here we show that overexpression of Bmi-1 enhances self-renewal of forebrain NSCs significantly more than those derived from spinal cord, demonstrating a regional difference in responsiveness. We show that forebrain NSCs require the forebrain-specific transcription factor Foxg1 for Bmi-1-dependent self-renewal, and that repression of p21 is a focus of this interaction. Bmi-1 enhancement of NSC self-renewal is significantly greater with increasing age and passage. Importantly, when Bmi-1 is overexpressed in cultured adult forebrain NSCs, they expand dramatically and continue to make neurons even after multiple passages, when control NSCs have become restricted to glial differentiation. Together these findings demonstrate the importance of Bmi-1 and Foxg1 cooperation to maintenance of NSC multipotency and self-renewal, and establish a useful method for generating abundant forebrain neurons ex vivo, outside the neurogenic niche.

Summary There is an emerging understanding of the importance of the vascular system within stem cell niches. Here, we examine whether neural stem cells (NSCs) in the adult subventricular zone (SVZ) lie close to blood vessels, using three-dimensional whole mounts, confocal microscopy, and automated computer-based image quantification. We found that the SVZ contains a rich plexus of blood vessels that snake along and within neuroblast chains. Cells expressing stem cell markers, including GFAP, and proliferation markers are closely apposed to the laminin-containing extracellular matrix (ECM) surrounding vascular endothelial cells. Apical GFAP+ cells are admixed within the ependymal layer and some span between the ventricle and blood vessels, occupying a specialized microenvironment. Adult SVZ progenitor cells express the laminin receptor α6β1 integrin, and blocking this inhibits their adhesion to endothelial cells, altering their position and proliferation in vivo, indicating that it plays a functional role in binding SVZ stem cells within the vascular niche.

The carotid body is an organ of the peripheral nervous system that senses oxygen concentration in the blood and responds to changes by regulating breathing. Pardal et al. (2007) now report the discovery of carotid body stem cells, which proliferate in response to hypoxia and generate neurons that secrete dopamine. This new source of adult stem cells may be useful in therapies for treating Parkinson's disease.

SummaryNeural progenitor cells (NPCs) in the adult subventricular zone (SVZ) are associated with ependymal and vasculature niches, which regulate stem cell self-renewal and differentiation. Activated Type B stem cells and their progeny, the transit-amplifying type C cells, which express EGFR, are most highly associated with vascular cells, indicating that this niche supports lineage progression. Here, we show that proliferative SVZ progenitor cells home to endothelial cells in a stromal-derived factor 1 (SDF1)- and CXC chemokine receptor 4 (CXCR4)-dependent manner. We show that SDF1 strongly upregulates EGFR and α6 integrin in activated type B and type C cells, enhancing their activated state and their ability to bind laminin in the vascular niche. SDF1 increases the motility of type A neuroblasts, which migrate from the SVZ toward the olfactory bulb. Thus, differential responses to SDF1 can regulate progenitor cell occupancy of and exit from the adult SVZ vascular niche.

SummaryNeurons arise in the adult forebrain subventricular zone (SVZ) from Type B neural stem cells (NSCs), raising considerable interest in the molecules that maintain this life-long neurogenic niche. Type B cells are anchored by specialized apical endfeet in the center of a pinwheel of ependymal cells. Here we show that the apical endfeet express high levels of the adhesion and signaling molecule vascular cell adhesion molecule-1 (VCAM1). Disruption of VCAM1 in vivo causes loss of the pinwheels, disrupted SVZ cytoarchitecture, proliferation and depletion of the normally quiescent apical Type B cells, and increased neurogenesis in the olfactory bulb, demonstrating a key role in niche structure and function. We show that VCAM1 signals via NOX2 production of reactive oxygen species (ROS) to maintain NSCs. VCAM1 on Type B cells is increased by IL-1β, demonstrating that it can act as an environmental sensor, responding to chemokines involved in tissue repair.

Results: We present an application that enables the quantitative analysis of multichannel 5-D (x, y, z, t, channel) and large montage confocal fluorescence microscopy images. The image sequences show stem cells together with blood vessels, enabling quantification of the dynamic behaviors of stem cells in relation to their vascular niche, with applications in developmental and cancer biology. Our application automatically segments, tracks, and lineages the image sequence data and then allows the user to view and edit the results of automated algorithms in a stereoscopic 3-D window while simultaneously viewing the stem cell lineage tree in a 2-D window. Using the GPU to store and render the image sequence data enables a hybrid computational approach. An inference-based approach utilizing user-provided edits to automatically correct related mistakes executes interactively on the system CPU while the GPU handles 3-D visualization tasks. Conclusions: By exploiting commodity computer gaming hardware, we have developed an application that can be run in the laboratory to facilitate rapid iteration through biological experiments. There is a pressing need for visualization and analysis tools for 5-D live cell image data. We combine accurate unsupervised processes with an intuitive visualization of the results. Our validation interface allows for each data set to be corrected to 100% accuracy, ensuring that downstream data analysis is accurate and verifiable. Our tool is the first to combine all of these aspects, leveraging the synergies obtained by utilizing validation information from stereo visualization to improve the low level image processing tasks., BioVis 2014 conference