The epigenetic factor Kmt2a/Mll1 regulates neural progenitor proliferation and neuronal and glial differentiation

Multiple epigenetic factors play a critical role in cell proliferation and differentiation. However, their function in embryogenesis, especially in neural development, is currently unclear. The Trithorax group (TrxG) homolog KMT2A (MLL1) is an important epigenetic regulator during development and has an especially well‐defined role in hematopoiesis. Translocation and aberrant expression of KMT2A is often observed in many tumors, indicating its proto‐oncogenic character. Here, we show that Kmt2a was essential for neural development in zebrafish embryos. Disrupting the expression of Kmt2a using morpholino antisense oligonucleotides and a dominant‐negative variant resulted in neurogenic phenotypes, including downregulated proliferation of neural progenitors, premature differentiation of neurons, and impaired gliogenesis. This study therefore revealed a novel function of Kmt2a in cell proliferation and differentiation, providing further insight into the function of TrxG proteins in neural development and brain tumors. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 452–462, 2015     

The chromatin modifying complex CoREST/LSD1 negatively regulates notch pathway during cerebral cortex development

The development of the cerebral cortex is a dynamic and coordinated process in which cell division, cell death, migration, and differentiation must be highly regulated to acquire the final architecture and functional competence of the mature organ. Notch pathway is an important regulator of differentiation and it is essential to maintain neural stem cell (NSC) pool. Here, we studied the role of epigenetic modulators such as lysine‐specific demethylase 1 (LSD1) and its interactor CoREST in the regulation of the Notch pathway activity during the development of the cerebral cortex. We found that CoREST and LSD1 interact in vitro with RBPJ‐κ in the repressor complex and these proteins are released upon overexpression of Notch intracellular domain (NICD). We corroborated LSD1 and RBPJ‐κ interaction in developing cerebral cortex and also found that LSD1 binds to the hes1 promoter. Knock‐down of CoREST and LSD1 by in utero electroporation increases Hes1 expression in vivo and decreases Ngn2. Interestingly, we found a functional interaction between CoREST and LSD1 with Notch pathway. This conclusion is based on the observation that both the defects in neuronal migration and the increase in the number of cells expressing Sox2 and Tbr2 were associated to the knock‐down of either CoREST or LSD1 and were reversed by the loss of Notch. These results demonstrate that CoREST and LSD1 downregulate the Notch pathway in the developing cerebral cortex, thus suggesting a role of epigenetic regulation in the fine tuning of cell differentiation. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1360–1373, 2016     

Trk signaling regulates neural precursor cell proliferation and differentiation during cortical development

Increasing evidence indicates that development of embryonic central nervous system precursors is tightly regulated by extrinsic cues located in the local environment. Here, we asked whether neurotrophin-mediated signaling through Trk tyrosine kinase receptors is important for embryonic cortical precursor cell development. These studies demonstrate that inhibition of TrkB (Ntrk2) and/or TrkC (Ntrk3) signaling using dominant-negative Trk receptors, or genetic knockdown of TrkB using shRNA, caused a decrease in embryonic precursor cell proliferation both in culture and in vivo. Inhibition of TrkB/C also caused a delay in the generation of neurons, but not astrocytes, and ultimately perturbed the postnatal localization of cortical neurons in vivo. Conversely, overexpression of BDNF in cortical precursors in vivo promoted proliferation and enhanced neurogenesis. Together, these results indicate that neurotrophin-mediated Trk signaling plays an essential, cell-autonomous role in regulating the proliferation and differentiation of embryonic cortical precursors and thus controls cortical development at earlier stages than previously thought.     

Histamine induces neural stem cell proliferation and neuronal differentiation by activation of distinct histamine receptors

Histamine has neurotransmitter/neuromodulator functions in the adult brain, but its role during CNS development has been elusive. We studied histamine effects on proliferation, cell death and differentiation of neuroepithelial stem cells from rat cerebral cortex in vitro . RT-PCR and Western blot experiments showed that proliferating and differentiated cells express histamine H₁, H₂ and H₃ receptors. Treatments with histamine concentrations (100 nM–1 mM) caused significant increases in cell numbers without affecting Nestin expression. Cell proliferation was evaluated by BrdU incorporation; histamine caused a significant increase dependent on H₂ receptor activation. Apoptotic cell death during proliferation was significantly decreased at all histamine concentrations, and cell death was promoted in a concentration-dependent manner by histamine in differentiated cells. Immunocytochemistry studies showed that histamine increased 3-fold the number of neurons after differentiation, mainly by activation of H₁ receptor, and also significantly decreased the glial (astrocytic) cell proportion, when compared to control conditions. In summary, histamine increases cell number during proliferative conditions, and has a neuronal-differentiating action on neural stem cells, suggesting that the elevated histamine concentration reported during development might play a role in cerebrocortical neurogenesis, by activation of H₂ receptors to promote proliferation of neural precursors, and favoring neuronal fate by H₁-mediated stimulation.     

Akhirin regulates the proliferation and differentiation of neural stem cells/progenitor cells at neurogenic niches in mouse brain

Specialized microenvironment, or neurogenic niche, in embryonic and postnatal mouse brain plays critical roles during neurogenesis throughout adulthood. The subventricular zone (SVZ) and the dentate gyrus (DG) of hippocampus in the mouse brain are two major neurogenic niches where neurogenesis is directed by numerous regulatory factors. Now, we report Akhirin (AKH), a stem cell maintenance factor in mouse spinal cord, plays a pivotal regulatory role in the SVZ and in the DG. AKH showed specific distribution during development in embryonic and postnatal neurogenic niches. Loss of AKH led to abnormal development of the ventricular zone and the DG along with reduction of cellular proliferation in both regions. In AKH knockout mice (AKH^−/−), quiescent neural stem cells (NSCs) increased, while proliferative NSCs or neural progenitor cells decreased at both neurogenic niches. In vitro NSC culture assay showed increased number of neurospheres and reduced neurogenesis in AKH^−/−. These results indicate that AKH, at the neurogenic niche, exerts dynamic regulatory role on NSC self-renewal, proliferation and differentiation during SVZ and hippocampal neurogenesis.     

Effects of sulforaphane on neural stem cell proliferation and differentiation

Sulforaphane (SFN) is a natural organosulfur compound with anti‐oxidant and anti‐inflammation properties. The objective of this study is to investigate the effect of SFN on the proliferation and differentiation of neural stem cells (NSC). NSCs were exposed to SFN at the concentrations ranging from 0.25 to 10 µM. Cell viability was evaluated with MTT assay and lactate dehydogenase (LDH) release assay. The proliferation of NSCs was evaluated with neurosphere formation assay and Ki‐67 staining. The level of Tuj‐1 was evaluated with immunostaining and Western blot to assess NSC neuronal differentiation. The expression of key proteins in the Wnt signaling pathway, including β‐catenin and cyclin D1, in response to SFN treatment or the Wnt inhibitor, DKK‐1, was determined by Western blotting. No significant cytotoxicity was seen for SFN on NSCs with SFN at concentrations of less than 10 µM. On the contrary, SFN of low concentrations stimulated cell proliferation and prominently increased neurosphere formation and NSC differentiation to neurons. SFN treatment upregulated Wnt signaling in the NSCs, whereas DKK‐1 attenuated the effects of SFN. SFN is a drug to promote NSC proliferation and neuronal differentiation when used at low concentrations. These protective effects are mediated by Wnt signaling pathway.     

Insulin-like growth factor-1 regulation of retinal progenitor cell proliferation and differentiation

Strategies to improve retinal progenitor cell (RPC) capacity to yield proliferative and multipotent pools of cells that can efficiently differentiate into retinal neurons, including photoreceptors, could be vital for cell therapy in retinal degenerative diseases. In this study, we found that insulin-like growth factor-1 (IGF-1) plays a role in the regulation of proliferation and differentiation of RPCs. Our results show that IGF-1 promotes RPC proliferation via IGF-1 receptors (IGF-1Rs), stimulating increased phosphorylation in the PI3K/Akt and MAPK/Erk pathways. An inhibitor experiment revealed that IGF-1-induced RPC proliferation was inhibited when the PI3K/Akt and MAPK/Erk pathways were blocked. Furthermore, under the condition of differentiation, IGF-1-pretreated RPCs prefer to differentiate into retinal neurons, including photoreceptors, in vitro, which is crucial for visual formation and visual restoration. These results demonstrate that IGF-1 accelerates the proliferation of RPCs and IGF-1 pretreated RPCs may have shown an increased potential for retinal neuron differentiation, providing a novel strategy for regulating the proliferation and differentiation of retinal progenitors in vitro and shedding light upon the application of RPCs in retinal cell therapy.     

Copine1 regulates neural stem cell functions during brain development

Copine 1 (CPNE1) is a well-known phospholipid binding protein in plasma membrane of various cell types. In brain cells, CPNE1 is closely associated with AKT signaling pathway, which is important for neural stem cell (NSC) functions during brain development. Here, we investigated the role of CPNE1 in the regulation of brain NSC functions during brain development and determined its underlying mechanism. In this study, abundant expression of CPNE1 was observed in neural lineage cells including NSCs and immature neurons in human. With mouse brain tissues in various developmental stages, we found that CPNE1 expression was higher at early embryonic stages compared to postnatal and adult stages. To model developing brain in vitro, we used primary NSCs derived from mouse embryonic hippocampus. Our in vitro study shows decreased proliferation and multi-lineage differentiation potential in CPNE1 deficient NSCs. Finally, we found that the deficiency of CPNE1 downregulated mTOR signaling in embryonic NSCs. These data demonstrate that CPNE1 plays a key role in the regulation of NSC functions through the activation of AKT-mTOR signaling pathway during brain development.     

Betacellulin regulates the proliferation and differentiation of retinal progenitor cells in vitro

Retinal progenitor cells (RPCs) hold great potential for the treatment of retinal degenerative diseases. However, their proliferation capacity and differentiation potential towards specific retinal neurons are limited, which limit their future clinical applications. Thus, it is important to improve the RPCs’ ability to proliferate and differentiate. Currently, epidermal growth factor (EGF) is commonly used to stimulate RPC growth in vitro. In this study, we find that betacellulin (BTC), a member of the EGF family, plays important roles in the proliferation and differentiation of RPCs. Our results showed that BTC can significantly promote the proliferation of RPCs more efficiently than EGF. EGF stimulated RPC proliferation through the EGFR/ErbB2‐Erk pathway, while BTC stimulated RPC proliferation more powerfully through the EGFR/ErbB2/ErbB4‐Akt/Erk pathway. Meanwhile, under differentiated conditions, the BTC‐pre‐treated RPCs were preferentially differentiated into retinal neurons, including photoreceptors, one of the most important types of cells for retinal cell replacement therapy, compared to the EGF‐pre‐treated RPCs. In addition, knockdown of endogenous BTC expression can also obviously promote RPC differentiation into retinal neuronal cells. This data demonstrate that BTC plays important roles in promoting RPC proliferation and differentiation into retinal neurons. This study may provide new insights into the study of RPC proliferation and differentiation and make a step towards the application of RPCs in the treatment of retinal degenerative diseases.     

Methylnitrosourea induces neural progenitor cell apoptosis and microcephaly in mouse embryos

BACKGROUND: Prenatal exposure to methylnitrosourea (MNU), an alkylating agent, induces microcephaly in mice. However, its pathogenetic mechanism has not been clarified, especially that in the development of the cerebral cortex. METHODS: ICR mice were treated with MNU at 10 mg/kg intraperitoneally on day 13.5 or 15.5 of gestation, and the embryos were observed histologically 24 hr after treatment with MNU or at term. To clarify the pathogenesis of microcephaly and histological changes, especially apoptosis, neurogenesis, and neural migration/positioning, we performed histological analysis employing a cell‐specific labeling experiment using thymidine‐like substances (BrdU, CldU, and IdU) and markers of neurons/neural stem cells. RESULTS: Histological abnormalities of the dorsal telencephalon, and the excessive cell death of proliferative neural progenitor/stem cells were noted in the MNU‐treated embryos. The highest frequencies of cell death occurred at 36 hr after MNU treatment, and little or no neurogenesis was observed in the ventricular zone of the dorsal telencephalon. Abnormality of the radial migration was caused by the reduction of radial fibers in the radial glias. Birth‐date analysis revealed the abnormal positioning of neurons and aberrant lamination of the cerebral cortex. CONCLUSIONS: Our data suggest that prenatal exposure to MNU induces the excessive cell death of neural precursor/stem cells, and the defective development of the cerebral cortex, resulting in microcephalic abnormalities. Birth Defects Res (Part B) 89:213–222, 2010. © 2010 Wiley‐Liss, Inc.     

PI3K/Akt and CREB regulate adult neural hippocampal progenitor proliferation and differentiation

The phosphoinositide 3-OH kinase (PI3K)/Akt pathway has been implicated in regulating several important cellular processes, including apoptosis, survival, proliferation, and metabolism. Using both pharmacological and genetic means, we demonstrate here that PI3K/Akt plays a crucial role in the proliferation of adult hippocampal neural progenitor cells. PI3K/Akt transduces intracellular signals from multiple mitogens, including basic fibroblast growth factor (FGF-2), Sonic hedgehog (Shh), and insulin-like growth factor 1 (IGF-1). In addition, retroviral vector-mediated over-expression of wild type Akt increased cell proliferation, while a dominant negative Akt inhibited proliferation. Furthermore, wild type Akt over-expression reduced glial (GFAP) and neuronal (beta-tubulin III) marker expression during differentiation, indicating that it inhibits cell differentiation. We also show that activation of the cAMP response element binding protein (CREB), which occurs in cells stimulated by FGF-2, is limited when Akt signaling is inhibited, demonstrating a link between Akt and CREB. Over-expression of wild type CREB increases progenitor proliferation, whereas dominant negative CREB only slightly decreases proliferation. These results indicate that PI3K/Akt signaling integrates extracellular signaling information to promote cellular proliferation and inhibit differentiation in adult neural progenitors.