Japanese encephalitis virus infects neural progenitor cells and decreases their proliferation

Japanese encephalitis virus (JEV), a common cause of encephalitis in humans, especially in children, leads to substantial neuronal injury. The survivors of JEV infection have severe cognitive impairment, motor and behavioral disorders. We hypothesize that depletion of neural progenitor cells (NPCs) by the virus culminates in neurological sequelae in survivors of Japanese encephalitis (JE). We utilized both in vivo model of JEV infection and in vitro neurosphere cultures to study progressive JEV infection. Cellular infection and cell death was determined by flow cytometry. BrdU administration in animals and in neurospheres was used to determine the proliferative ability of NPCs. JEV leads to massive loss of actively proliferating NPC population from the subventricular zone (SVZ). The ability of JEV infected subventricular zone cells to form neurospheres is severely compromised. This can be attributed to JEV infection in NPCs, which however do not result in robust death of the resilient NPC cells. Instead, JEV suppresses the cycling ability of these cells, preventing their proliferation. JEV primarily targets at a critical postnatal age and severely diminishes the NPC pool in SVZ, thus impairing the process of recovery after the insult. This arrested growth and proliferation of NPCs might have an effect on the neurological consequences in JE survivors.     

Down-regulation of WNK1 protein kinase in neural progenitor cells suppresses cell proliferation and migration

WNK1, a Ser/Thr protein kinase, is widely expressed in many tissues. Its biological functions are largely unknown. Disruption of the WNK1 gene in mice leads to embryonic lethality at day 13, implicating a critical role of WNK1 in embryonic development. To investigate this potential function, we used antisense strategy to knock down the expression of WNK1 in a mouse neural progenitor cell line, C17.2. Down-regulation of WNK1 in C17.2 cells greatly reduced cell growth. Addition of epidermal growth factor (EGF), a mitogen for C17.2 cells, had no effect on growth. The WNK1-knockdown cells showed a flat and rounded morphology, characteristic of the immature and non-differentiated phenotype of the progenitor cells; this was further demonstrated by immunostaining for the progenitor and neuronal markers. Migration of the WNK1-knockdown C17.2 cells was reduced as tested in culture dishes or Matrigel-covered chambers. Moreover, activation of extracellular signal-regulated kinase (ERK1)/2 and ERK5 by EGF in the WNK1-knockdown cells was suppressed. These results demonstrate a novel function of WNK1 in proliferation, migration, and differentiation of neural progenitor cells, likely by mechanisms involving activation of the mitogen-activated protein (MAP) kinase ERK1/2 and/or ERK5 pathways.     

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.     

Elimination of the geomagnetic field stimulates the proliferation of mouse neural progenitor and stem cells

Living organisms are exposed to the geomagnetic field (GMF) throughout their lifespan. Elimination of the GMF, resulting in a hypogeomagnetic field (HMF), leads to central nervous system dysfunction and abnormal development in animals. However, the cellular mechanisms underlying these effects have not been identified so far. Here, we show that exposure to an HMF (<200 nT), produced by a magnetic field shielding chamber, promotes the proliferation of neural progenitor/stem cells (NPCs/NSCs) from C57BL/6 mice. Following seven-day HMF-exposure, the primary neurospheres (NSs) were significantly larger in size, and twice more NPCs/NSCs were harvested from neonatal NSs, when compared to the GMF controls. The self-renewal capacity and multipotency of the NSs were maintained, as HMF-exposed NSs were positive for NSC markers (Nestin and Sox2), and could differentiate into neurons and astrocyte/glial cells and be passaged continuously. In addition, adult mice exposed to the HMF for one month were observed to have a greater number of proliferative cells in the subventricular zone. These findings indicate that continuous HMF-exposure increases the proliferation of NPCs/NSCs,in vitro and in vivo. HMF-disturbed NPCs/ NSCs production probably Affects brain development and function, which provides a novel clue for elucidating the cellular mechanisms of the bio-HMF response.     

Differentiating embryonic neural progenitor cells induce blood-brain barrier properties

The blood-brain barrier (BBB) is a multifunctional endothelial interface separating the bloodstream from the brain interior. Although the mature BBB is well characterized, the embryonic development of this complex system remains poorly understood. Embryonic neural progenitor cells (NPC) are a potential inductive cell type populating the developing brain, and their ability to influence BBB properties was therefore examined. When puromycin-purified brain microvascular endothelial cells (BMEC) were co-cultured with embryonic NPC in a two-compartment Transwell system, the BMEC exhibited enhanced barrier properties in the form of increased transendothelial electrical resistance (TEER) and decreased permeability to the small molecule tracer, sodium fluorescein. These changes required the presence of NPC in the early stages of differentiation and were accompanied by alterations in the fidelity of BMEC tight junctions as indicated by occludin, claudin 5, and zonula occluden-1 redistribution at cell-cell borders. In contrast to the findings with NPC, post-natal astrocytes elicited a delayed, but longer duration response in BMEC TEER. BMEC co-culture also suppressed neuronal differentiation of NPC indicating a reciprocal signaling between the two cell populations. This study demonstrates that NPC-BMEC interactions are prevalent and for the first time demonstrates that NPC are capable of inducing BBB properties.     

Inhibition of the proliferation of cultured immortalized schwann cells by forskolin with a decreased basal level of diacylglycerol

The repetitive passages of a Schwann cell culture results in the appearance of immortalized cells. In order to investigate the direct effects of cyclic AMP (cAMP) on Schwann cell proliferation, we used the immortalized Schwann cells because the responses of a short-term Schwann cell culture to agents increasing the intracellular cAMP are more complicated and it does not seem that all of them are due to the direct effects of cAMP. By adding up to 200 M of forskolin, an adenylate cyclase activator, to the culture medium, Schwann cell proliferation was inhibited and the intracellular 1,2-diacylglycerol (DG) level was decreased in a dose-dependent manner to 44 and 53% of the control values, respectively. The protein phosphorylation activity in the cytosol from the cell treated with 100 M forskolin, assayed with myelin basic protein as the acceptor, decreased to 78% and this inhibition was then reversed by the addition of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeable DG, to the assay mixture. The cell proliferation inhibited by forskolin was also restored by the addition of OAG. These data suggest that cAMP inhibits both the activity of protein kinase C (PKC) and consequently cell proliferation through suppression of intracellular DG level, an activator of PKC. Since the inositol 1,4,5-triphosphate level and the hydrolysis of phosphatidylcholine to DG and phosphorylcholine were not affected, forskolin therefore appears to suppress the de novo synthesis of DG.     

GSK3 inhibitors stabilize Wee1 and reduce cerebellar granule cell progenitor proliferation

Ubiquitin mediated proteolysis is required for transition from one cell cycle phase to another. For instance, the mitosis inhibitor Wee1 is targeted for degradation during S phase and G2 to allow mitotic entry. Wee1 is an essential tyrosine kinase required for the G2/M transition and S-phase progression. Although several studies have concentrated on Wee1 regulation during mitosis, few have elucidated its degradation during interphase. Our prior studies have demonstrated that Wee1 is degraded via CK1δ dependent phosphorylation during the S and G2/M phases of the cell cycle. Here we demonstrate that GSK3β may work in concert with CK1δ to induce Wee1 destruction during interphase. We generated small molecules that specifically stabilized Wee1. We profiled these compounds against 296 kinases and found that they inhibit GSK3α and GSK3β, suggesting that Wee1 may be targeted for proteolysis by GSK3. Consistent with this notion, known GSK3 inhibitors stabilized Wee1 and GSK3β depletion reduced Wee1 turnover. Given Wee1''s central role in cell cycle progression, we predicted that GSK3 inhibitors should limit cell proliferation. Indeed, we demonstrate that GSK3 inhibitors potently inhibited proliferation of the most abundant cell in the mammalian brain, the cerebellar granule cell progenitor (GCP). These studies identify a previously unappreciated role for GSK3β mediated regulation of Wee1 during the cell cycle and in neurogenesis. Furthermore, they suggest that pharmacological inhibition of Wee1 may be therapeutically attractive in some cancers where GSK-3β or Wee1 are dysregulated.     

GSK3 inhibitors stabilize Wee1 and reduce cerebellar granule cell progenitor proliferation

Ubiquitin mediated proteolysis is required for transition from one cell cycle phase to another. For instance, the mitosis inhibitor Wee1 is targeted for degradation during S phase and G2 to allow mitotic entry. Wee1 is an essential tyrosine kinase required for the G2/M transition and S-phase progression. Although several studies have concentrated on Wee1 regulation during mitosis, few have elucidated its degradation during interphase. Our prior studies have demonstrated that Wee1 is degraded via CK1δ dependent phosphorylation during the S and G2/M phases of the cell cycle. Here we demonstrate that GSK3β may work in concert with CK1δ to induce Wee1 destruction during interphase. We generated small molecules that specifically stabilized Wee1. We profiled these compounds against 296 kinases and found that they inhibit GSK3α and GSK3β, suggesting that Wee1 may be targeted for proteolysis by GSK3. Consistent with this notion, known GSK3 inhibitors stabilized Wee1 and GSK3β depletion reduced Wee1 turnover. Given Wee1's central role in cell cycle progression, we predicted that GSK3 inhibitors should limit cell proliferation. Indeed, we demonstrate that GSK3 inhibitors potently inhibited proliferation of the most abundant cell in the mammalian brain, the cerebellar granule cell progenitor (GCP). These studies identify a previously unappreciated role for GSK3β mediated regulation of Wee1 during the cell cycle and in neurogenesis. Furthermore, they suggest that pharmacological inhibition of Wee1 may be therapeutically attractive in some cancers where GSK-3β or Wee1 are dysregulated.     

D1‐like dopamine receptors regulate GABAA receptor function to modulate hippocampal neural progenitor cell proliferation

The proliferation and differentiation of neural progenitor (NP) cells can be regulated by neurotransmitters including GABA and dopamine. The present study aimed to examine how these two neurotransmitter systems interact to affect post‐natal hippocampal NP cell proliferation in vitro. Mouse hippocampal NP cells express functional GABA_A receptors, which upon activation led to an increase in intracellular calcium levels via the opening of L‐type calcium channels. Activation of these GABA_A receptors also caused a significant decrease in proliferation; an effect that required the entry of calcium through L‐type calcium channels. Furthermore, while activation of D₁‐like dopamine receptors had no effect on proliferation, it abrogated the suppressive effects of GABA_A receptor activation on proliferation. The effects of D₁‐like dopamine receptors are associated with a decrease in the ability of GABA_A receptors to increase intracellular calcium levels, and a reduction in the surface expression of GABA_A receptors. In this way, D₁‐like dopamine receptor activation can increase the proliferation of NP cells by preventing GABA_A receptor‐mediated inhibition of proliferation. These results suggest that, in conditions where NP cell proliferation is under the tonic suppression of GABA, agonists which act through D₁‐like dopamine receptors may increase the proliferation of neural progenitors.     

ZDHHC16 modulates FGF/ERK dependent proliferation of neural stem/progenitor cells in the zebrafish telencephalon

In vertebrates, neural stem/progenitor cells (NSPCs) maintenance is critical for nervous system development and homeostasis. However, the molecular mechanisms underlying the maintenance of NSPCs have not been fully elucidated. Here, we demonstrated that zebrafish ZDHHC16, a DHHC encoding protein, which was related to protein palmitoylation after translation, was expressed in the developing forebrain, and especially in the telencephalon. Loss‐ and gain‐of‐function studies showed that ZDHHC16 played a crucial role in the regualtion of NSPCs proliferation during zebrafish telencephalic development, via a mechanism dependent on its palmitoyltransferase activity. Further analyses showed that the inhibition of ZDHHC16 led to inactivation of the FGF/ERK signaling pathway during telencephalic NSPCs proliferation and maintenance. Taken together, our results suggest that ZDHHC16 activity is essential for early NSPCs proliferation where it acts to activate the FGF/ERK network, allowing for the initiation of proliferation –regulated gene expression programs. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1014–1028, 2016     

Neuregulin induces proliferation of neural progenitor cells via PLC/PKC pathway

Nestin-expressing neural progenitor cells (NPCs) have been isolated from hippocampus of brains and propagated with epidermal growth factor and basic fibroblast growth factor (bFGF). However, the underlying signaling mechanisms regulating NPC proliferation remain elusive. Here we showed that neuregulinbeta1 (NRG), like bFGF, effectively promoted the proliferation of hippocampus-derived NPCs and maintained the progenitor states of NPCs. Activation of protein kinase C (PKC), a downstream effector of phospholipase C (PLC), with 12-O-tetradecanoylphorbol-13-acetate (TPA) mimicked the NRG-induced proliferation of NPCs. The synergic effect of TPA plus NRG on neurosphere growth further prompted us to find that NRG induced NPC propagation through PLC/PKC signaling pathway. ErbB4, an important functional receptor of NRG, had an interaction with PLCgamma1 protein. In addition, inactivation of PLC pathway led to severe proliferative suppression of NPCs. Our study suggests that activation of PLC/PKC pathway plays an essential role in the NRG-induced proliferation of hippocampus-derived NPCs.     

细胞外信号调节激酶在小鼠神经干细胞增殖中的作用

本文旨在探讨细胞外信号调节激酶(extracellular signal-regulated kinase 1/2, ERK1/2)对小鼠神经干细胞增殖的影响.分离E14.5小鼠皮层神经干细胞,通过Western blot检测神经干细胞增殖过程中磷酸化ERK1/2的表达情况,以及不同浓度PD98059处理对神经干细胞ERK1/2磷酸化及神经球形成的影响,并用CCK-8法检测PD98059对神经干细胞增殖的影响.结果显示:ERK1/2在体外培养的神经下细胞增殖过程中被激活;PD98059显著抑制ERK1/2磷酸化及神经干细胞的成球率,且存在剂量效应依赖关系;加入PD98059后神经干细胞的生长被抑制.以上结果表明,ERK1/2在小鼠神经干细胞增殖中具有重要的作用,阻断ERK1/2信号通路后可抑制神经干细胞的增殖.     

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.