Clock genes regulate neurogenic transcription factors,including NeuroD1, and the neuronal differentiation of adult neural stem/progenitor cells

The circadian clock system plays multiple roles in our bodies, and clock genes are expressed in various brain regions, including the lateral subventricular zone (SVZ) where neural stem/progenitor cells (NSPCs) persist and postnatal neurogenesis continues. However, the functions of clock genes in adult NSPCs are not well understood. Here, we first investigated the expression patterns of Clock and Bmal1 in the SVZ by immunohistochemistry and then verified how the expression levels of 17 clock and clock-related genes changed during differentiation of cultured adult NSPCs using quantitative RT-PCR. Finally, we used RNAi to observe the effects of Clock and Bmal1 on neuronal differentiation. Our results revealed that Clock and Bmal1 were expressed in the SVZ and double-stained with the neural progenitor marker Nestin and neural stem marker GFAP. In cultured adult NSPCs, the clock genes changed their expression patterns during differentiation, and interestingly, Bmal1 started endogenous oscillation. Moreover, gene silencing of Clock or Bmal1 by RNAi decreased the percentages of neuronal marker Map2-positive cells and expression levels of NeuroD1 mRNA. These findings suggest that clock genes are involved in the neuronal differentiation of adult NSPCs and may extend our understanding of various neurological/psychological disorders linked to adult neurogenesis and circadian rhythm.     

Transcriptional Profiling of Adult Neural Stem-Like Cells from the Human Brain

There is a great potential for the development of new cell replacement strategies based on adult human neural stem-like cells. However, little is known about the hierarchy of cells and the unique molecular properties of stem- and progenitor cells of the nervous system. Stem cells from the adult human brain can be propagated and expanded in vitro as free floating neurospheres that are capable of self-renewal and differentiation into all three cell types of the central nervous system. Here we report the first global gene expression study of adult human neural stem-like cells originating from five human subventricular zone biopsies (mean age 42, range 33–60). Compared to adult human brain tissue, we identified 1,189 genes that were significantly up- and down-regulated in adult human neural stem-like cells (1% false discovery rate). We found that adult human neural stem-like cells express stem cell markers and have reduced levels of markers that are typical of the mature cells in the nervous system. We report that the genes being highly expressed in adult human neural stem-like cells are associated with developmental processes and the extracellular region of the cell. The calcium signaling pathway and neuroactive ligand-receptor interactions are enriched among the most differentially regulated genes between adult human neural stem-like cells and adult human brain tissue. We confirmed the expression of 10 of the most up-regulated genes in adult human neural stem-like cells in an additional sample set that included adult human neural stem-like cells (n = 6), foetal human neural stem cells (n = 1) and human brain tissues (n = 12). The NGFR, SLITRK6 and KCNS3 receptors were further investigated by immunofluorescence and shown to be heterogeneously expressed in spheres. These receptors could potentially serve as new markers for the identification and characterisation of neural stem- and progenitor cells or as targets for manipulation of cellular fate.     

Neural Stem/Progenitor Cells Derived from the Embryonic Dorsal Telencephalon of D6/GFP Mice Differentiate Primarily into Neurons After Transplantation into a Cortical Lesion

D6 is a promoter/enhancer of the mDach1 gene that is involved in the development of the neocortex and hippocampus. It is expressed by proliferating neural stem/progenitor cells (NSPCs) of the cortex at early stages of neurogenesis. The differentiation potential of NSPCs isolated from embryonic day 12 mouse embryos, in which the expression of green fluorescent protein (GFP) is driven by the D6 promoter/enhancer, has been studied in vitro and after transplantation into the intact adult rat brain as well as into the site of a photochemical lesion. The electrophysiological properties of D6/GFP-derived cells were studied using the whole-cell patch-clamp technique, and immunohistochemical analyses were carried out. D6/GFP-derived neurospheres expressed markers of radial glia and gave rise predominantly to immature neurons and GFAP-positive cells during in vitro differentiation. One week after transplantation into the intact brain or into the site of a photochemical lesion, transplanted cells expressed only neuronal markers. D6/GFP-derived neurons were characterised by the expression of tetrodotoxin-sensitive Na⁺-currents and K _A- and K _DR currents sensitive to 4-aminopyridine. They were able to fire repetitive action potentials and responded to the application of GABA. Our results indicate that after transplantation into the site of a photochemical lesion, D6/GFP-derived NSPCs survive and differentiate into neurons, and their membrane properties are comparable to those transplanted into the non-injured cortex. Therefore, region-specific D6/GFP-derived NSPCs represent a promising tool for studying neurogenesis and cell replacement in a damaged cellular environment.     

The role of the N-methyl-d-aspartate receptor in the proliferation of adult hippocampal neural stem and precursor cells

New neurons are continuously generated from resident pools of neural stem and precursor cells(NSPCs)in the adult brain.There are multiple pathways through which adult neurogenesis is regulated,and here we review the role of the N-methyl-D-aspartate receptor(NMDAR)in regulating the proliferation of NSPCs in the adult hippocampus.Hippocampal-dependent learning tasks,enriched environments,running,and activity-dependent synaptic plasticity,all potently up-regulate hippocampal NSPC proliferation.We first consider the requirement of the NMDAR in activity-dependent synaptic plasticity,and the role the induction of synaptic plasticity has in regulating NSPCs and newborn neurons.We address how specific NMDAR agonists and antagonists modulate proliferation,both in vivo and in vitro,and then review the evidence supporting the hypothesis that NMDARs are present on NSPCs.We believe it is important to understand the mechanisms underlying the activation of adult neurogenesis,given the potential that endogenous stem cell populations have for repopulating the hippocampus with functional new neurons.In conditions such as age-related memory decline,neurodegeneration and psychiatric disease,mature neurons are lost or become defective;as such,stimulating adult neurogenesis may provide a therapeutic strategy to overcome these conditions.     

Neural stem/progenitor cells display a low requirement for oxidative metabolism independent of hypoxia inducible factor‐1alpha expression

Neural stem/progenitor cells (NSPCs) are multipotent cells within the embryonic and adult brain that give rise to both neuronal and glial cell lineages. Maintenance of NSPC multipotency is promoted by low oxygen tension, although the metabolic underpinnings of this trait have not been described. In this study, we investigated the metabolic state of undifferentiated NSPCs in culture, and tested their relative reliance on oxidative versus glycolytic metabolism for survival, as well as their dependence on hypoxia inducible factor‐1alpha (HIF‐1α) expression for maintenance of metabolic phenotype. Unlike primary neurons, NSPCs from embryonic and adult mice survived prolonged hypoxia in culture. In addition, NSPCs displayed greater susceptibility to glycolytic inhibition compared with primary neurons, even in the presence of alternative mitochondrial TCA substrates. NSPCs were also more resistant than neurons to mitochondrial cyanide toxicity, less capable of utilizing galactose as an alternative substrate to glucose, and more susceptible to pharmacological inhibition of the pentose phosphate pathway by 6‐aminonicotinamide. Inducible deletion of exon 1 of the Hif1a gene improved the ability of NSPCs to utilize pyruvate during glycolytic inhibition, but did not alter other parameters of metabolism, including their ability to withstand prolonged hypoxia. Taken together, these data indicate that NSPCs have a relatively low requirement for oxidative metabolism for their survival and that hypoxic resistance is not dependent upon HIF‐1α signaling.     

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.     

Microglial p38α MAPK is critical for LPS-induced neuron degeneration, through a mechanism involving TNFα

Background

The identification of factors that compromise neurogenesis is aimed at improving stem cell-based approaches in the field of regenerative medicine. Interferon gamma (IFNγ) is a main pro-inflammatory cytokine and up-regulated during several neurological diseases. IFNγ is generally thought to beneficially enhance neurogenesis from fetal or adult neural stem/precursor cells (NSPCs).

Results

We now provide direct evidence to the contrary that IFNγ induces a dysfunctional stage in a substantial portion of NSPC-derived progeny in vitro characterized by simultaneous expression of glial fibrillary acid protein (GFAP) and neuronal markers, an abnormal gene expression and a functional phenotype neither typical for neurons nor for mature astrocytes. Dysfunctional development of NSPCs under the influence of IFNγ was finally demonstrated by applying the microelectrode array technology. IFNγ exposure of NSPCs during an initial 7-day proliferation period prevented the subsequent adequate differentiation and formation of functional neuronal networks.

Conclusions

Our results show that immunocytochemical analyses of NSPC-derived progeny are not necessarily indicating the correct cellular phenotype specifically under inflammatory conditions and that simultaneous expression of neuronal and glial markers rather point to cellular dysregulation. We hypothesize that inhibiting the impact of IFNγ on NSPCs during neurological diseases might contribute to effective neurogenesis and regeneration.     

Not(ch) just development: Notch signalling in the adult brain

The Notch pathway is often regarded as a developmental pathway, but components of Notch signalling are expressed and active in the adult brain. With the advent of more sophisticated genetic manipulations, evidence has emerged that suggests both conserved and novel roles for Notch signalling in the adult brain. Not surprisingly, Notch is a key regulator of adult neural stem cells, but it is increasingly clear that Notch signalling also has roles in the regulation of migration, morphology, synaptic plasticity and survival of immature and mature neurons. Understanding the many functions of Notch signalling in the adult brain, and its dysfunction in neurodegenerative disease and malignancy, is crucial to the development of new therapeutics that are centred around this pathway.     

ARF6 activated by the LHCG receptor through the cytohesin family of guanine nucleotide exchange factors mediates the receptor internalization and signaling

The luteinizing hormone chorionic gonadotropin receptor (LHCGR) is a G(s)-coupled GPCR that is essential for the maturation and function of the ovary and testis. LHCGR is internalized following its activation, which regulates the biological responsiveness of the receptor. Previous studies indicated that ADP-ribosylation factor (ARF)6 and its GTP-exchange factor (GEF) cytohesin 2 regulate LHCGR internalization in follicular membranes. However, the mechanisms by which ARF6 and cytohesin 2 regulate LHCGR internalization remain incompletely understood. Here we investigated the role of the ARF6 signaling pathway in the internalization of heterologously expressed human LHCGR (HLHCGR) in intact cells using a combination of pharmacological inhibitors, siRNA and the expression of mutant proteins. We found that human CG (HCG)-induced HLHCGR internalization, cAMP accumulation and ARF6 activation were inhibited by Gallein (βγ inhibitor), Wortmannin (PI 3-kinase inhibitor), SecinH3 (cytohesin ARF GEF inhibitor), QS11 (an ARF GAP inhibitor), an ARF6 inhibitory peptide and ARF6 siRNA. However, Dynasore (dynamin inhibitor), the dominant negative mutants of NM23-H1 (dynamin activator) and clathrin, and PBP10 (PtdIns 4,5-P2-binding peptide) inhibited agonist-induced HLHCGR and cAMP accumulation but not ARF6 activation. These results indicate that heterotrimeric G-protein, phosphatidylinositol (PI) 3-kinase (PI3K), cytohesin ARF GEF and ARF GAP function upstream of ARF6 whereas dynamin and clathrin act downstream of ARF6 in the regulation of HCG-induced HLHCGR internalization and signaling. In conclusion, we have identified the components and molecular details of the ARF6 signaling pathway required for agonist-induced HLHCGR internalization.     

Radmis,a Novel Mitotic Spindle Protein that Functions in Cell Division of Neural Progenitors

Developmental dynamics of neural stem/progenitor cells (NSPCs) are crucial for embryonic and adult neurogenesis, but its regulatory factors are not fully understood. By differential subtractive screening with NSPCs versus their differentiated progenies, we identified the radmis (radial fiber and mitotic spindle)/ckap2l gene, a novel microtubule-associated protein (MAP) enriched in NSPCs. Radmis is a putative substrate for the E3-ubiquitin ligase, anaphase promoting complex/cyclosome (APC/C), and is degraded via the KEN box. Radmis was highly expressed in regions of active neurogenesis throughout life, and its distribution was dynamically regulated during NSPC division. In embryonic and perinatal brains, radmis localized to bipolar mitotic spindles and radial fibers (basal processes) of dividing NSPCs. As central nervous system development proceeded, radmis expression was lost in most brain regions, except for several neurogenic regions. In adult brain, radmis expression persisted in the mitotic spindles of both slowly-dividing stem cells and rapid amplifying progenitors. Overexpression of radmis in vitro induced hyper-stabilization of microtubules, severe defects in mitotic spindle formation, and mitotic arrest. In vivo gain-of-function using in utero electroporation revealed that radmis directed a reduction in NSPC proliferation and a concomitant increase in cell cycle exit, causing a reduction in the Tbr2-positive basal progenitor population and shrinkage of the embryonic subventricular zone. Besides, radmis loss-of-function by shRNAs induced the multipolar mitotic spindle structure, accompanied with the catastrophe of chromosome segregation including the long chromosome bridge between two separating daughter nuclei. These findings uncover the indispensable role of radmis in mitotic spindle formation and cell-cycle progression of NSPCs.     

Region-specific Differentiation Potential of Adult Rat Spinal Cord Neural Stem/Precursors and Their Plasticity in Response to In Vitro Manipulation

This study characterized the differentiation of neural stem/precursor cells (NSPCs) isolated from different levels of the spinal cord (cervical vs lumbar cord) and different regions along the neuraxis (brain vs cervical spinal cord) of adult male Wistar enhanced green fluorescent protein rats. The differentiation of cervical spinal cord NSPCs was further examined after variation of time in culture, addition of growth factors, and changes in cell matrix and serum concentration. Brain NSPCs did not differ from cervical cord NSPCs in the percentages of neurons, astrocytes, or oligodendrocytes but produced 26.9% less radial glia. Lumbar cord NSPCs produced 30.8% fewer radial glia and 6.9% more neurons compared with cervical cord NSPCs. Spinal cord NSPC differentiation was amenable to manipulation by growth factors and changes in in vitro conditions. This is the first study to directly compare the effect of growth factors, culturing time, serum concentration, and cell matrix on rat spinal cord NSPCs isolated, propagated, and differentiated under identical conditions. (J Histochem Cytochem 57:405–423, 2009)     

Brain from bone: Efficient "meta-differentiation" of marrow stroma-derived mature osteoblasts to neurons with Noggin or a demethylating agent

Bone marrow stromal cells are able to differentiate into adipogenic, chondrogenic, myogenic, osteogenic, and cardiomyogenic lineages, all of which are limited to a mesoderm-derived origin. In this study, we showed that neurons, which are of an ectoderm-origin, could be generated from marrow-derived stromal cells by specific inducers, fibronectin/ornithine coating, and neurosphere formation. The neurons generated from marrow stroma formed neurites, expressed neuron-specific markers and genes, and started to respond to depolarizing stimuli as functional mature neurons. Among stromal cells, isolated mature osteoblasts which had strong in vivo osteogenic activity could be efficiently converted into functional neurons. This transdifferentiation or meta-differentiation was enhanced by Noggin, an inhibitor of bone morphogenetic proteins, in comparison with 5-azacytidine, a demethylating agent capable of altering the gene expression pattern. Marrow stroma is therefore a potential source of cells for neural cell transplantation.     

KIAA0649, a 1A6/DRIM-interacting protein with the oncogenic potential

We identified a 1A6/DRIM (down-regulated in metastasis) interacting protein, KIAA0649 during the yeast two-hybrid screen. The interaction between KIAA0649 and 1A6/DRIM was further confirmed by GST-pull-down and co-immunoprecipitation assays. KIAA0649 was originally identified from human brain tissue. However, its biological function remains unknown. In this report, we showed that KIAA0649 mRNA is widely expressed in human multiple tissues and cell lines. We have also demonstrated that KIAA0649 has oncogenic characteristics: it enhances colony formation, allows anchorage-independent growth. Moreover, KIAA0649 exogenous expression in NIH3T3 fibroblasts caused tumor development in nude mice.     

Brorin, a novel secreted bone morphogenetic protein antagonist, promotes neurogenesis in mouse neural precursor cells

We identified a gene encoding a novel secreted protein in mice and humans and named it Brorin. Mouse Brorin consists of 324 amino acids with a putative secreted signal sequence at its amino terminus and two cysteine-rich domains in its core region. Positions of 10 cysteine residues in the domains of Brorin are similar to those in the cysteine-rich domains of members of the Chordin family. However, the amino acid sequence of Brorin is not significantly similar to that of any other member of the Chordin family, indicating that Brorin is a unique member of the family. Mouse Brorin protein produced in cultured cells was efficiently secreted into the culture medium. The protein inhibited the activity of bone morphogenetic protein 2 (BMP2) and BMP6 in mouse preosteoblastic MC3T3-E1 cells. Mouse Brorin was predominantly expressed in neural tissues in embryos and also predominantly expressed in the adult brain. In the brain, the expression was detected in neurons, but not glial cells. The neural tissue-specific expression profile of Brorin is quite distinct from that of any other member of the Chordin family. Brorin protein promoted neurogenesis, but not astrogenesis, in mouse neural precursor cells. The present findings indicate that Brorin is a novel secreted BMP antagonist that potentially plays roles in neural development and functions.