Search

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.

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.

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., (©2019 American Association for Cancer Research.)

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., (© 2019 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

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)., (© 2019 Velasco et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The vascular compartment of the adult brain ventricular-subventricular zone (V-SVZ) is a critical regulator of neural stem cell and progenitor function. Blood enters the V-SVZ via arteries and arterioles to capillaries that then connect with venules and veins to return blood to the heart. We found that stromal cell-derived factor 1 (SDF1) is expressed by a subpopulation of V-SVZ vessels, the capillaries, and that actively proliferating neural stem cells (NSCs) and progenitors are preferentially associated with these SDF1-positive vessels. In contrast, slowly dividing or quiescent NSCs are most prevalent near SDF1-negative vessels. By conditional knockout, we found that loss of SDF1 signaling in NSCs stimulates lineage progression and NSC displacement from the vessel niche. With aging, SDF1/CXCR4 signaling is dysregulated, coincident with reduced proliferation and increased displacement of dividing cells from the vasculature. Our findings demonstrate SDF1-based vascular heterogeneity in the niche and suggest that reduced SDF1 signaling contributes to age-related declines in adult neurogenesis., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Neural stem cells (NSCs) persist throughout life in the dentate gyrus and the ventricular-subventricular zone, where they continuously provide new neurons and some glia. These cells are found in specialized niches that regulate quiescence, activation, differentiation, and cell fate choice. A key aspect of the regulatory niche is the vascular plexus, which modulates NSC behavior during tissue homeostasis and regeneration. During aging, NSCs become depleted and dysfunctional, resulting in reduced neurogenesis and poor brain repair. In this review, we discuss the emerging evidence that changes in the vascular niche both structurally and functionally contribute to reduced neurogenesis during aging and how this might contribute to reduced plasticity and repair in the aged brain., (Copyright © 2017 the American Physiological Society.)

The ventricular-subventricular zone harbors neural stem cells (NSCs) that can differentiate into neurons, astrocytes, and oligodendrocytes. This process requires loss of stem cell properties and gain of characteristics associated with differentiated cells. miRNAs function as important drivers of this transition; miR-124, -128, and -137 are among the most relevant ones and have been shown to share commonalities and act as proneurogenic regulators. We conducted biological and genomic analyses to dissect their target repertoire during neurogenesis and tested the hypothesis that they act cooperatively to promote differentiation. To map their target genes, we transfected NSCs with antagomiRs and analyzed differences in their mRNA profile throughout differentiation with respect to controls. This strategy led to the identification of 910 targets for miR-124, 216 for miR-128, and 652 for miR-137. The target sets show extensive overlap. Inspection by gene ontology and network analysis indicated that transcription factors are a major component of these miRNAs target sets. Moreover, several of these transcription factors form a highly interconnected network. Sp1 was determined to be the main node of this network and was further investigated. Our data suggest that miR-124, -128, and -137 act synergistically to regulate Sp1 expression. Sp1 levels are dramatically reduced as cells differentiate and silencing of its expression reduced neuronal production and affected NSC viability and proliferation. In summary, our results show that miRNAs can act cooperatively and synergistically to regulate complex biological processes like neurogenesis and that transcription factors are heavily targeted to branch out their regulatory effect., (© 2015 AlphaMed Press.)

Background: Neural stem cells are motile and proliferative cells that undergo mitosis, dividing to produce daughter cells and ultimately generating differentiated neurons and glia. Understanding the mechanisms controlling neural stem cell proliferation and differentiation will play a key role in the emerging fields of regenerative medicine and cancer therapeutics. Stem cell studies in vitro from 2-D image data are well established. Visualizing and analyzing large three dimensional images of intact tissue is a challenging task. It becomes more difficult as the dimensionality of the image data increases to include time and additional fluorescence channels. There is a pressing need for 5-D image analysis and visualization tools to study cellular dynamics in the intact niche and to quantify the role that environmental factors play in determining cell fate., Results: We present an application that integrates visualization and quantitative analysis of 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. We combine unsupervised image analysis algorithms with an interactive 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.

Neurons 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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Neural 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 alpha6 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., (Copyright 2010 Elsevier Inc. All rights reserved.)

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.

Brain development was thought to be largely hardwired and accomplished by birth, and the brain was thought to have essentially no regenerative capacity. The remarkable discovery of adult neurogenesis and neural stem cells (NSCs) existing in the mature CNS changed that, allowing us to think optimistically about CNS repair. These discoveries helped to generate a robust field of neural progenitor cell biology, with relevance to CNS development, pathogenesis, the search for novel neurological therapies, as well as our understanding of how the brain works.

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 alpha6beta1 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.