[Show abstract][Hide abstract] ABSTRACT: A single asymmetric division by an adult neural stem cell (NSC) ultimately generates dozens of differentiated progeny, a feat made possible by the proliferative expansion of transit-amplifying progenitor cells (TAPs). Although NSC activation and TAP expansion is determined by pro- and anti-proliferative signals found within the niche, remarkably little is known about how these cells integrate simultaneous conflicting signals. We investigated this question focusing on the subventricular zone (SVZ) niche of the adult murine forebrain. Using primary cultures of SVZ cells, we demonstrate that Epidermal Growth Factor (EGF) and Bone Morphogenetic Protein (BMP)-2 are particularly powerful pro- and anti-proliferative factors for SVZ-derived neural precursors. Dose-response experiments showed that when simultaneously exposed to both signals, BMP dominantly suppressed EGF-induced proliferation; moreover, this dominance extended to all parameters of neural precursor behavior tested, including inhibition of proliferation, modulation of cell cycle, promotion of differentiation, and increase of cell death. BMP's anti-proliferative effect did not involve inhibition of mTORC1 or ERK signaling, key mediators of EGF-induced proliferation, and had distinct stage-specific consequences, promoting TAP differentiation but NSC quiescence. In line with these in vitro data, in vivo experiments showed that exogenous BMP limits EGF-induced proliferation of TAPs while inhibition of BMP-SMAD signaling promotes activation of quiescent NSCs. These findings clarify the stage-specific effects of BMPs on SVZ neural precursors, and support a hierarchical model in which the anti-proliferative effects of BMP dominate over EGF proliferation signaling to constitutively drive TAP differentiation and NSC quiescence.
Preview · Article · Oct 2015 · Frontiers in Neuroscience
[Show abstract][Hide abstract] ABSTRACT: The adult mammalian spinal cord has limited regenerative capacity in settings such as spinal cord injury (SCI) and multiple sclerosis (MS). Recent studies have revealed that ependymal cells lining the central canal possess latent neural stem cell potential, undergoing proliferation and multi-lineage differentiation following experimental SCI. To determine whether reactive ependymal cells are a realistic endogenous cell population to target in order to promote spinal cord repair, we assessed the spatiotemporal dynamics of ependymal cell proliferation for up to 35 days in three models of spinal pathologies: contusion SCI using the Infinite Horizon impactor, focal demyelination by intraspinal injection of lysophosphatidylcholine (LPC), and autoimmune-mediated multi-focal demyelination using the active experimental autoimmune encephalomyelitis (EAE) model of MS. Contusion SCI at the T9-10 thoracic level stimulated a robust, long-lasting and long-distance wave of ependymal proliferation that peaked at 3 days in the lesion segment, 14 days in the rostral segment, and was still detectable at the cervical level, where it peaked at 21 days. This proliferative wave was suppressed distal to the contusion. Unlike SCI, neither chemical- nor autoimmune-mediated demyelination triggered ependymal cell proliferation at any time point, despite the occurrence of demyelination (LPC and EAE), remyelination (LPC) and significant locomotor defects (EAE). Thus, traumatic SCI induces widespread and enduring activation of reactive ependymal cells, identifying them as a robust cell population to target for therapeutic manipulation after contusion; conversely, neither demyelination, remyelination nor autoimmunity appears sufficient to trigger proliferation of quiescent ependymal cells in models of MS-like demyelinating diseases.
[Show abstract][Hide abstract] ABSTRACT: Environmental enrichment (EE) exerts powerful effects on brain physiology, and is widely used as an experimental and therapeutic tool. Typical EE paradigms are multifactorial, incorporating elements of physical exercise, environmental complexity, social interactions and stress, however the specific contributions of these variables have not been separable using conventional housing paradigms. Here, we evaluated the impacts of these individual variables on adult hippocampal neurogenesis by using a novel "Alternating EE" paradigm. For 4 weeks, adult male CD1 mice were alternated daily between two enriched environments; by comparing groups that differed in one of their two environments, the individual and combinatorial effects of EE variables could be resolved. The Alternating EE paradigm revealed that (1) voluntary running for 3 days/week was sufficient to increase both mitotic and post-mitotic stages of hippocampal neurogenesis, confirming the central importance of exercise; (2) a complex environment (comprised of both social interactions and rotated inanimate objects) had no effect on neurogenesis itself, but enhanced depolarization-induced c-Fos expression (attributable to social interactions) and buffered stress-induced plasma corticosterone levels (attributable to inanimate objects); and (3) neither social isolation, group housing, nor chronically increased levels of plasma corticosterone had a prolonged impact on neurogenesis. Mouse strain, handling and type of running apparatus were tested and excluded as potential confounding factors. These findings provide valuable insights into the relative effects of key EE variables on adult neurogenesis, and this "Alternating EE" paradigm represents a useful tool for exploring the contributions of individual EE variables to mechanisms of neural plasticity.
[Show abstract][Hide abstract] ABSTRACT: In the brains of adult vertebrates, including humans, neurogenesis occurs in restricted niches where it maintains cellular turnover and cognitive plasticity. In virtually all species, however, aging is associated with a significant decline in adult neurogenesis. Moreover, an acceleration of neurogenic defects is observed in models of Alzheimer's disease and other neurodegenerative diseases, suggesting an involvement in aging- and disease-associated cognitive deficits. To gain insights into when, how and why adult neurogenesis decreases in the aging brain, we critically reviewed the scientific literature on aging of the rodent subventricular zone, the neurogenic niche of the adult forebrain. Our analysis revealed that deficits in the neurogenic pathway are largely established by middle age, but that there remains striking ambiguity in the underlying mechanisms, especially at the level of stem and progenitor cells. We identify and discuss several challenging issues that have contributed to these key gaps in our current knowledge. In the future, addressing these issues should help untangle the interactions between neurogenesis, aging and aging-associated diseases.
Full-text · Article · Jun 2013 · European Journal of Neuroscience
[Show abstract][Hide abstract] ABSTRACT: Adult forebrain neurogenesis is dynamically regulated. Multiple families of niche-derived cues have been implicated in this regulation, but the precise roles of key intracellular signaling pathways remain vaguely defined. Here, we show that mammalian target of rapamycin (mTOR) signaling is pivotal in determining proliferation versus quiescence in the adult forebrain neural stem cell (NSC) niche. Within this niche, mTOR complex-1 (mTORC1) activation displays stage specificity, occurring in transiently amplifying (TA) progenitor cells but not in GFAP+ stem cells. Inhibiting mTORC1 depletes the TA progenitor pool in vivo and suppresses epidermal growth factor (EGF)-induced proliferation within neurosphere cultures. Interestingly, mTORC1 inhibition induces a quiescence-like phenotype that is reversible. Likewise, mTORC1 activity and progenitor proliferation decline within the quiescent NSC niche of the aging brain, while EGF administration reactivates the quiescent niche in an mTORC1-dependent manner. These findings establish fundamental links between mTOR signaling, proliferation, and aging-associated quiescence in the adult forebrain NSC niche.
No preview · Article · Oct 2012 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
[Show abstract][Hide abstract] ABSTRACT: Neuroblastoma (NB) is the most common and lethal extracranial solid tumor of childhood. Despite aggressive therapy, more than half of the children with advanced NB will die of uncontrolled metastatic disease. After chemotherapy, tumor-initiating cells (TICs) could persist, cause relapses and metastasis. The aim of this study is to demonstrate the tumor-initiating properties of CD133high NB cells and to identify new specific genetic abnormalities. Isolation of the CD133high cell population from NB cell lines was followed by neurosphere formation, soft agar assays, and orthotopic injections in NOD/SCID/IL2Rγc-null mice. A differential genotyping analysis was performed with Affymetrix SNP 6.0 arrays on CD133low and CD133high populations and the frequency of the abnormalities of 36 NB tumors was determined. Our results show that CD133high NB cells possess tumor-initiating properties, as CD133high cells formed significantly more neurospheres and produced significantly more colonies in soft agar than CD133low. Injection of 500 CD133high cells was sufficient to generate primary tumors and frequent metastases in mice. Differential genotyping analysis demonstrated two common regions with gains (16p13.3 and 19p13.3) including the gene EFNA2 in the CD133high population, and two with loss of heterozygosity (16q12.1 and 21q21.3) in the CD133low population. The gain of EFNA2 correlated with increased expression of the corresponding protein. These abnormalities were found in NB samples and some were significantly correlated with CD133 expression. Our results show that CD133high NB cells have TICs properties and present different genotyping characteristics compared to CD133low cells. Our findings reveal insights into new therapeutic targets in NB TICs.
Full-text · Article · Aug 2012 · Genes Chromosomes and Cancer
[Show abstract][Hide abstract] ABSTRACT: Hippocampal neurogenesis continues into adulthood in mammalian vertebrates, and in experimental rodent models it is powerfully stimulated by exposure to a voluntary running wheel. In this study, we demonstrate that exposure to a running wheel environment, in the absence of running, is sufficient to regulate specific aspects of hippocampal neurogenesis. Adult mice were provided with standard housing, housing enriched with a running wheel or housing enriched with a locked wheel (i.e., an environment comparable to that of running animals, without the possibility of engaging in running). We found that mice in the running wheel and locked wheel groups exhibited equivalent increases in proliferation within the neurogenic niche of the dentate gyrus; this included comparable increases in the proliferation of radial glia-like stem cells and the number of proliferating neuroblasts. However, only running animals displayed increased numbers of postmitotic neuroblasts and mature neurons. These results demonstrate that the running wheel environment itself is sufficient for promoting proliferation of early lineage hippocampal precursors, while running per se enables newly generated neuroblasts to survive and mature into functional hippocampal neurons. Thus, both running-independent and running-dependent stimuli are integral to running wheel-induced hippocampal neurogenesis.
[Show abstract][Hide abstract] ABSTRACT: Stem cells can exist in either active or quiescent states. In the aging hippocampus, adult neural stem cells (aNSCs) shift into a quiescent state, contributing to age-related reductions in hippocampal neurogenesis. Here, we focused on the subventricular zone (SVZ) stem cell niche of the adult forebrain, asking to what extent quiescence-associated changes in aNSCs are initiated between early and middle-age. Immunohistochemical and label retention experiments revealed that the overall output of the SVZ stem cell system was already highly decreased in middle-aged mice (12-months-old) compared with young adult mice (2-month-old), as measured by reduced marker expression for multiple neural precursor sub-populations and diminished addition of SVZ-derived neuroblasts to the olfactory bulbs (OBs). These changes were associated with significant cytological aberrations within the SVZ niche, including an overall atrophy of the SVZ and accumulation of large lipid droplets within ependymal cells, which are key support cells of the SVZ niche. Importantly, the reduced output of the middle-aged SVZ stem cell system correlated with quiescence-associated changes in middle-aged aNSCs. Specifically, while tissue culture experiments showed that young adult and middle-aged forebrains possessed equal numbers of neurosphere-forming aNSCs, the middle-aged neurospheres exhibited differences in their in vitro properties, and middle-aged aNSCs in vivo divided less frequently. These findings demonstrate that aNSCs begin undergoing quiescence-associated changes between early and mid-adulthood in the mouse SVZ, and serve as a useful framework for further studies aimed at defining the early events involved in aging-associated quiescence of aNSCs.
[Show abstract][Hide abstract] ABSTRACT: Alzheimer's disease (AD) affects cognitive modalities that are known to be regulated by adult neurogenesis, such as hippocampal- and olfactory-dependent learning and memory. However, the relationship between AD-associated pathologies and alterations in adult neurogenesis has remained contentious. In the present study, we performed a detailed investigation of adult neurogenesis in the triple transgenic (3xTg) mouse model of AD, a unique model that generates both amyloid plaques and neurofibrillary tangles, the hallmark pathologies of AD. In both neurogenic niches of the brain, the hippocampal dentate gyrus and forebrain subventricular zone, we found that 3xTg mice had decreased numbers of (i) proliferating cells, (ii) early lineage neural progenitors, and (iii) neuroblasts at middle age (11months old) and old age (18months old). These decreases correlated with major reductions in the addition of new neurons to the respective target areas, the dentate granule cell layer and olfactory bulb. Within the subventricular zone niche, cytological alterations were observed that included a selective loss of subependymal cells and the development of large lipid droplets within the ependyma of 3xTg mice, indicative of metabolic changes. Temporally, there was a marked acceleration of age-related decreases in 3xTg mice, which affected multiple stages of neurogenesis and was clearly apparent prior to the development of amyloid plaques or neurofibrillary tangles. Our findings indicate that AD-associated mutations suppress neurogenesis early during disease development. This suggests that deficits in adult neurogenesis may mediate premature cognitive decline in AD.
No preview · Article · Sep 2010 · European Journal of Neuroscience
[Show abstract][Hide abstract] ABSTRACT: Voluntary wheel-running induces a rapid increase in proliferation and neurogenesis by neural precursors present in the adult rodent hippocampus. In contrast, the responses of hippocampal and other central nervous system neural precursors following longer periods of voluntary physical activity are unclear and are an issue of potential relevance to physical rehabilitation programs. We investigated the effects of a prolonged, 6-week voluntary wheel-running paradigm on neural precursors of the CD1 mouse hippocampus and forebrain. Examination of the hippocampus following 6 weeks of running revealed two to three times as many newly born neurons and 60% more proliferating cells when compared with standard-housed control mice. Among running mice, the number of newly born neurons correlated with the total running distance. To establish the effects of wheel-running on hippocampal precursors dividing during later stages of the prolonged running regime, BrdU was administered after 3 weeks of running and the BrdU-retaining cells were analyzed 18 days later. Quantifications revealed that the effects of wheel-running were maintained in late-stage proliferating cells, as running mice had two to three times as many BrdU-retaining cells within the hippocampal dentate gyrus, and these yielded greater proportions of both mature neurons and proliferative cells. The effects of prolonged wheel-running were also detected beyond the hippocampus. Unlike short-term wheel-running, prolonged wheel-running was associated with higher numbers of proliferating cells within the ventral forebrain subventricular region, a site of age-associated decreases in neural precursor proliferation and neurogenesis. Collectively, these findings indicate that (i) prolonged voluntary wheel-running maintains an increased level of hippocampal neurogenesis whose magnitude is linked to total running performance, and (ii) that it influences multiple neural precursor populations of the adult mouse brain.
[Show abstract][Hide abstract] ABSTRACT: A stem cell's microenvironment, or "niche," is a critical regulator of its behaviour. In the adult mammalian spinal cord, central canal ependymal cells possess latent neural stem cell properties, but the ependymal cell niche has not yet been described. Here, we identify important similarities and differences between the central canal ependymal zone and the forebrain subventricular zone (SVZ), a well-characterized niche of neural stem cells. First, direct immunohistochemical comparison of the spinal cord ependymal zone and the forebrain SVZ revealed distinct patterns of neural precursor marker expression. In particular, ependymal cells in the spinal cord were found to be bordered by a previously uncharacterized sub-ependymal layer, which is relatively less elaborate than that of the SVZ and comprised of small numbers of astrocytes, oligodendrocyte progenitors and neurons. Cell proliferation surrounding the central canal occurs in close association with blood vessels, but unlike in the SVZ, involves mainly ependymal rather than sub-ependymal cells. These proliferating ependymal cells typically self-renew rather than produce transit-amplifying progenitors, as they generate doublets of progeny that remain within the ependymal layer and show no evidence of a lineage relationship to sub-ependymal cells. Interestingly, the dorsal pole of the central canal was found to possess a sub-population of tanycyte-like cells that express markers of both ependymal cells and neural precursors, and their presence correlates with higher numbers of dorsally proliferating ependymal cells. Together, these data identify key features of the spinal cord ependymal cell niche, and suggest that dorsal ependymal cells possess the potential for stem cell activity. This work provides a foundation for future studies aimed at understanding ependymal cell regulation under normal and pathological conditions.
[Show abstract][Hide abstract] ABSTRACT: The isolation and experimental manipulation of multipotent precursors is of increasing therapeutic relevance. We recently reported the generation of cultures of Skin-derived Precursors ('SKPs'), multipotent cells that can be isolated from the dermis of embryonic, neonatal, and adult rodent skin (1), and from adult human skin (2) SKPs have similarities to stem cells of the embryonic neural crest (3), and differentiate into a variety of neural and mesodermal cell phenotypes, including peripheral neurons and glial cells, smooth muscle cells, bone, cartilage, and adipocytes (3-5). Here, we detail the establishment, propagation, neural differentiation, and immunocytochemical analysis of SKP cultures.
No preview · Article · Feb 2009 · Methods in Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Nestin-expressing cells were identified in the normal rat heart characterized by a small cell body and numerous processes and following an ischemic insult migrated to the infarct region. The present study was undertaken to identify the phenotype, origin and biological role of nestin-expressing cells during reparative fibrosis. A neural stem cell phenotype was identified based on musashi-1 expression, growth as a neurosphere, and differentiation to a neuronal cell. Using the Wnt1-cre; Z/EG transgenic mouse model, which expresses EGFP in embryologically-derived neural crest cells, the reporter signal was detected in nestin-expressing cells residing in the heart. In infarcted human hearts, nestin-expressing cells were detected in the viable myocardium and the scar and morphologically analogous to the population identified in the rat heart. Following either an ischemic insult or the acute administration of 6-hydroxydopamine, sympathetic sprouting was dependent on the physical association of neurofilament-M immunoreactive fibres with nestin-positive processes emanating from neural stem cells. To specifically study the biological role of the subpopulation in the infarct region, neural stem cells were isolated from the scar, fluorescently labelled and transplanted in the heart of 3-day post-MI rats. Injected scar-derived neural stem cells migrated to the infarct region and were used as a substrate for de novo blood vessel formation. These data have demonstrated that the heart contains a resident population of neural stem cells derived from the neural crest and participate in reparative fibrosis. Their manipulation could provide an alternative approach to ameliorate the healing process following ischemic injury.
No preview · Article · Aug 2008 · Journal of Molecular and Cellular Cardiology
[Show abstract][Hide abstract] ABSTRACT: We previously made the surprising finding that cultures of multipotent precursors can be grown from the dermis of neonatal and adult mammalian skin. These skin-derived precursors (SKPs) display multi-lineage differentiation potential, producing both neural and mesodermal progeny in vitro, and are an apparently novel precursor cell type that is distinct from other known precursors within the skin. In this review, we begin by placing these findings within the context of the rapidly evolving stem cell field. We then describe our recent efforts focused on understanding the developmental biology of SKPs, discussing the idea that SKPs are neural crest-related precursors that (i) migrate into the skin during embryogenesis, (ii) persist within a specific dermal niche, and (iii) play a key role in the normal physiology, and potentially pathology, of the skin. We conclude by highlighting some of the therapeutic implications and unresolved questions raised by these studies.
Preview · Article · Feb 2008 · Philosophical Transactions of The Royal Society B Biological Sciences
[Show abstract][Hide abstract] ABSTRACT: Multipotent precursors similar to stem cells of the embryonic neural crest (NC) have been identified in several postnatal tissues, and are potentially useful for research and therapeutic purposes. However, their neurogenic potential, including their ability to produce electrophysiologically active neurons, is largely unexplored. We investigated this issue with regard to skin-derived precursors (SKPs), multipotent NC-related precursors isolated from the dermis of skin. SKP cultures follow an appropriate pattern and time-course of neuronal differentiation, with proliferating nestin-expressing SKPs generating post-mitotic neuronal cells that co-express pan-neuronal and peripheral autonomic lineage markers. These SKP-derived neuron-like cells survive and maintain their peripheral phenotype for at least 5 weeks when transplanted into the CNS environment of normal or kainate-injured hippocampal slices. Undifferentiated SKPs retain key neural precursor properties after multi-passage expansion, including growth factor dependence, nestin expression, neurogenic potential, and responsiveness to embryonic neural crest fate determinants. Despite undergoing an apparently appropriate neurogenic process, however, SKP-derived neuron-like cells possess an immature electrophysiological profile. These findings indicate that SKPs retain latent neurogenic properties after residing in a non-neural tissue, but that additional measures will be necessary to promote their differentiation into electrophysiologically active neurons.
No preview · Article · Oct 2006 · Experimental Neurology
[Show abstract][Hide abstract] ABSTRACT: Precursor cells of the embryonic cortex sequentially generate neurons and then glial cells, but the mechanisms regulating this neurogenic-to-gliogenic transition are unclear. Using cortical precursor cultures, which temporally mimic this in vivo differentiation pattern, we demonstrate that cortical neurons synthesize and secrete the neurotrophic cytokine cardiotrophin-1, which activates the gp130-JAK-STAT pathway and is essential for the timed genesis of astrocytes in vitro. Our data indicate that a similar phenomenon also occurs in vivo. In utero electroporation of neurotrophic cytokines in the environment of embryonic cortical precursors causes premature gliogenesis, while acute perturbation of gp130 in cortical precursors delays the normal timed appearance of astrocytes. Moreover, the neonatal cardiotrophin-1-/- cortex contains fewer astrocytes. Together, these results describe a neural feedback mechanism; newly born neurons produce cardiotrophin-1, which instructs multipotent cortical precursors to generate astrocytes, thereby ensuring that gliogenesis does not occur until neurogenesis is largely complete.