Connexin 36 expression regulates neuronal differentiation from neural progenitor cells

Background

Gap junction communication has been shown in glial and neuronal cells and it is thought they mediate inter- and intra-cellular communication. Connexin 36 (Cx36) is expressed extensively in the developing brain, with levels peaking at P14 after which its levels fall and its expression becomes entirely neuronal. These and other data have led to the hypothesis that Cx36 may direct neuronal coupling and neurogenesis during development.

Methodology/Principal Findings

To investigate Cx36 function we used a neurosphere model of neuronal cell development and developed lentiviral Cx36 knockdown and overexpression strategies. Cx36 knockdown was confirmed by western blotting, immunocytochemistry and functionally by fluorescence recovery after photobleaching (FRAP). We found that knockdown of Cx36 in neurosphere neuronal precursors significantly reduced neuronal coupling and the number of differentiated neurons. Correspondingly, the lentiviral mediated overexpression of Cx36 significantly increased the number of neurons derived from the transduced neurospheres. The number of oligodendrocytes was also significantly increased following transduction with Cx36 indicating they may support neuronal differentiation.

Conclusions/Significance

Our data suggests that astrocytic and neuronal differentiation during development are governed by mechanisms that include the differential expression of Cx36.     

SEPT7 regulates Ca2+ entry through Orai channels in human neural progenitor cells and neurons

Human neural progenitor cells (hNPCs) are self-renewing cells of neural lineage that can be differentiated into neurons of different subtypes. Here we show that SEPT7, a member of the family of filament-forming GTPases called septins, prevents constitutive Ca²⁺ entry through the store-operated Ca²⁺ entry channel, Orai in hNPCs and in differentiated neurons and is thus required for neuronal calcium homeostasis. Previous work in Drosophila neurons has shown that loss of one copy of the evolutionarily-conserved dSEPT7 gene leads to elevated Ca²⁺ entry via Orai, in the absence of ER-Ca²⁺ store depletion. We have identified an N-terminal polybasic region of SEPT7, known to interact with membrane-localized phospholipids, as essential for spontaneous calcium entry through Orai in hNPCs, whereas the GTPase domain of dSEPT7 is dispensable for this purpose. Re-organisation of Orai1 and the ER-Ca²⁺ sensor STIM1 observed near the plasma membrane in SEPT7 KD hNPCs, supports the idea that Septin7 containing heteromers prevent Ca²⁺ entry through a fraction of STIM-Orai complexes. Possible mechanisms by which SEPT7 reduction leads to opening of Orai channels in the absence of store-depletion are discussed.     

Transforming growth factor-beta1 regulates the fate of cultured spinal cord-derived neural progenitor cells

Abstract. Objectives: We have evaluated the physiological roles of transforming growth factor‐β1 (TGF‐β1) on differentiation, migration, proliferation and anti‐apoptosis characteristics of cultured spinal cord‐derived neural progenitor cells. Methods: We have used neural progenitor cells that had been isolated and cultured from mouse spinal cord tissue, and we also assessed the relevant reaction mechanisms using an activin‐like kinase (ALK)‐specific inhibitory system including an inhibitory RNA, and found that it involved potential signalling molecules such as phosphatidylinositol‐3‐OH kinase (PI3K)/Akt and mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK1/2). Results and Conclusions: Transforming growth factor‐β1‐mediated cell population growth was activated after treatment and was also effectively blocked by an ALK41517‐synthetic inhibitor (4‐(5‐benzo(1,3) dioxol‐5‐yl‐4‐pyridine‐2‐yl‐1H‐imidazole‐2‐yl) benzamide (SB431542) and ALK siRNA, thereby indicating the involvement of SMAD2 in the TGF‐β1‐mediated growth and migration of these neural progenitors cells (NPC). In the present study, TGF‐β1 actively induced NPC migration in vitro. Furthermore, TGF‐β1 demonstrated extreme anti‐apoptotic behaviour against hydrogen peroxide‐mediated apoptotic cell death. At low dosages, TGF‐β1 enhanced (by approximately 76%) cell survival against hydrogen peroxide treatment via inactivation of caspase‐3 and ‐9. TGF‐β1‐treated NPCs down‐regulated Bax expression and cytochrome c release; in addition, the cells showed up‐regulated Bcl‐2 and thioredoxin reductase 1. They also had increased p38, Akt and ERK1/2 phosphorylation, showing the involvement of both the PI3K/Akt and MAPK/ERK1/2 pathways in the neuroprotective effects of TGF‐β1. Interestingly, these effects operate on specific subtypes of cells, including neurones, neural progenitor cells and astrocytes in cultured spinal cord tissue‐derived cells. Lesion sites of spinal cord‐overexpressing TGF‐β1‐mediated prevention of cell death, cell growth and migration enhancement activity have been introduced as a possible new basis for therapeutic strategy in treatment of neurodegenerative disorders, including spinal cord injuries.     

Autophagy regulates biliary differentiation of hepatic progenitor cells through Notch1 signaling pathway

Autophagy plays important roles in self-renewal and differentiation of stem cells. Hepatic progenitor cells (HPCs) are thought to have the ability of self-renewal as well as possess a bipotential capacity, which allows them to differentiate into both hepatocytes and bile ductular cells. However, how autophagy contributes to self-renewal and differentiation of hepatic progenitor cells is not well understood. In this study, we use a well-established rat hepatic progenitor cell lines called WB-F344, which is treated with 3.75 mM sodium butyrate (SB) to promote the differentiation of WB-F344 along the biliary phenotype. We found that autophagy was decreased in the early stage of biliary differentiation, and maintained a low level at the late stage. Activation of autophagy by rapamycin or starvation suppressed the biliary differentiation of WB-F344. Further study reported that autophagy inhibited Notch1 signaling pathway, which contributed to biliary differentiation and morphogenesis. In conclusions, autophagy regulates biliary differentiation of hepatic progenitor cells through Notch1 signaling pathway.     

TRIM32-dependent transcription in adult neural progenitor cells regulates neuronal differentiation

In the adult mammalian brain, neural stem cells in the subventricular zone continuously generate new neurons for the olfactory bulb. Cell fate commitment in these adult neural stem cells is regulated by cell fate-determining proteins. Here, we show that the cell fate-determinant TRIM32 is upregulated during differentiation of adult neural stem cells into olfactory bulb neurons. We further demonstrate that TRIM32 is necessary for the correct induction of neuronal differentiation in these cells. In the absence of TRIM32, neuroblasts differentiate slower and show gene expression profiles that are characteristic of immature cells. Interestingly, TRIM32 deficiency induces more neural progenitor cell proliferation and less cell death. Both effects accumulate in an overproduction of adult-generated olfactory bulb neurons of TRIM32 knockout mice. These results highlight the function of the cell fate-determinant TRIM32 for a balanced activity of the adult neurogenesis process.     

Angiogenic activity of endothelial progenitor cells through angiopoietin-1 and angiopoietin-2

Angiogenesis is a regulated process involving the proliferation, migration, and remodeling of different cell types particularly mature endothelial cells and recently discovered progenitor cells, named as endothelial progenitor cells (EPCs). Up to now, many attempts have been made to understand the dynamic balance of pro- and anti-angiogenic factors on EPCs on different milieu. It has been accepted that Ang-1, -2 and Tie-1, -2 signaling play a key role on angiogenesis pathways in endothelial lineage cells. In the current experiment, the angiogenic/angio-modulatory potency of Ang-1 and -2 was investigated on isolated EPCs. Freshly isolated EPCs were exposed to different concentrations of Ang-1 and -2 (25 and 50?ng/ml) over a course of 7 and 14 days. Corroborating to our results, a superior effect of Ang-1 on angiogenic properties, including an increased concentration of vascular endothelial growth factor, in vitro tubulogenesis, EPC migratory, Tie-2 expression and clonogenicity, was determined. A large amount of positive mature endothelium markers was achieved in EPCs being-exposed to Ang-1 peptide. Nonetheless, the number of CD133 positive cells increased in the presence of Ang-2. Collectively, we conclude that Ang-1 potentially induces functional and mature vascular-like behavior in EPCs more than Ang-2.     

Gi2 signaling enhances proliferation of neural progenitor cells in the developing brain

Our previous study showed that the pertussis toxin-sensitive G protein, Gi2, is selectively localized in the ventricular zone of embryonic brains, where the neuroepithelial cells undergo active proliferation. In order to clarify the role of Gi2 in this site, we first administered pertussis toxin by an exo-utero manipulation method into the lateral ventricle of mouse brain at embryonic day 14.5. Examination at embryonic day 18.5 revealed that pertussis toxin-injected embryos had brains with thinner cerebral cortices, made up of fewer constituent cells. Bromodeoxyuridine labeling revealed fewer numbers of bromodeoxyuridine-positive cells in the cerebral cortices of pertussis toxin-injected embryos, suggesting impaired proliferation of neuroepithelial cells. Next we cultured neural progenitor cells from rat embryonic brains and evaluated the mitogenic effects of agonists for several Gi-coupled receptors that are known to be expressed in the ventricular zone. Among agonists tested, endothelin most effectively stimulated the incorporation of [3H]thymidine in the presence of fibronectin, via the endothelin-B receptor. This was associated with phosphorylation of extracellular signal-regulated kinase, and pertussis toxin partially inhibited both endothelin-stimulated DNA synthesis and phosphorylation of extracellular signal-regulated kinase. Injection of endothelin-3 into the ventricle of embryonic brains increased numbers of bromodeoxyuridine-positive cells in the cerebral cortex, whereas injection of an endothelin-B receptor antagonist decreased them. These findings indicate that Gi2 mediates signaling from receptors such as the endothelin-B receptor to maintain mitogenic activity in the neural progenitor cells of developing brain.     

c-kit marks late retinal progenitor cells and regulates their differentiation in developing mouse retina

Retinal progenitor cells are believed to display altered proliferation and differentiation during retinal development, suggesting that retinal progenitor cell populations are not homogeneous. However, the composition of progenitor cell populations is not known, due in part to the lack of known surface markers identifying distinct stages of retinal progenitor cells. We found a dramatic change in the expression profile of the cell surface antigens c-kit and stage-specific embryonic antigen-1 (SSEA-1) in retinal progenitor cells during development. While SSEA-1 was expressed early in development, c-kit expression peaked in late stage progenitor cells. The identification of these developmental markers enabled us to characterize distinct sub-populations of retinal progenitor cells. Progenitor cell subpopulations expressing either SSEA-1, c-kit, or both showed different proliferation and differentiation abilities. Although SSEA-1-positive cells were augmented by beta-catenin signaling, c-kit-positive cells were positively regulated by Notch signaling. Taken together, our data suggest that c-kit and SSEA-1 can be used to spatiotemporally differentiate retinal progenitor populations that have intrinsically distinct characteristics. Prolonged expression of c-kit by a retrovirus resulted in the promotion of proliferation and the appearance of nestin-positive cells in the presence of the c-kit ligand, stem cell factor (SCF). This suggests a role for c-kit, Notch, and the beta-catenin signaling network in retinal development.     

Ectopic expression of angiopoietin-1 promotes neuronal differentiation in neural progenitor cells through the Akt pathway

In regions of adult neurogenesis, neural progenitor cells (NPCs) are found in close proximity to blood vessels within a so-called ‘vascular niche’. Neurogenesis is linked to angiogenesis via certain growth factors. We propose that angiopoietin-1 (Ang1), which is similar to VEGF, has a unique role in neurogenesis independent of its role in angiogenesis. In this study, primary cultures of NPCs were transduced with recombinant adenoviruses expressing Ang1 and induced to differentiate with dibutyryl cyclic AMP (dbcAMP). Neuronal differentiation was evaluated by quantitative PCR, immunofluorescence microscopy and Western blot analysis. The results show that ectopic expression of Ang1 promotes neuronal differentiation and neurite outgrowth in NPCs, while this effect was blocked by the presence of anti-Tie2 receptor antibody or the PI3-K inhibitor, LY294002. Our results suggest that Ang1, identified originally as an angiogenic factor, can also stimulate in vitro neurogenesis in NPCs through the Akt pathway.     

Dystroglycan regulates structure, proliferation and differentiation of neuroepithelial cells in the developing vertebrate CNS

In the developing CNS alpha- and beta-dystroglycan are highly concentrated in the endfeet of radial neuroepithelial cells at the contact site to the basal lamina. We show that injection of anti-dystroglycan Fab fragments, knockdown of dystroglycan using RNAi, and overexpression of a dominant-negative dystroglycan protein by microelectroporation in neuroepithelial cells of the chick retina and optic tectum in vivo leads to the loss of their radial morphology, to hyperproliferation, to an increased number of postmitotic neurons, and to an altered distribution of several basally concentrated proteins. Moreover, these treatments also altered the oriented growth of axons from retinal ganglion cells and from tectal projection neurons. In contrast, expression of non-cleavable dystroglycan protein in neuroepithelial cells reduced their proliferation and their differentiation to postmitotic neurons. These results demonstrate that dystroglycan plays a key role in maintaining neuroepithelial cell morphology, and that interfering with dystroglycan function influences proliferation and differentiation of neuroepithelial cells. These data also suggest that an impaired dystroglycan function in neuroepithelial cells might be responsible for some of the severe brain abnormalities observed in certain forms of congenital muscular dystrophy.     

Snf2l regulates Foxg1-dependent progenitor cell expansion in the developing brain

Balancing progenitor cell self-renewal and differentiation is essential for brain development and is regulated by the activity of chromatin remodeling complexes. Nevertheless, linking chromatin changes to specific pathways that control cortical histogenesis remains a challenge. Here we identify a genetic interaction between the chromatin remodeler Snf2l and Foxg1, a key regulator of neurogenesis. Snf2l mutant mice exhibit forebrain hypercellularity arising from increased Foxg1 expression, increased progenitor cell expansion, and delayed differentiation. We demonstrate that Snf2l binds to the Foxg1 locus at midneurogenesis and that the phenotype is rescued by reducing Foxg1 dosage, thus revealing that Snf2l and Foxg1 function antagonistically to regulate brain size.     

Numb regulates the balance between Notch recycling and late-endosome targeting in Drosophila neural progenitor cells

The Notch signaling pathway plays essential roles in both animal development and human disease. Regulation of Notch receptor levels in membrane compartments has been shown to affect signaling in a variety of contexts. Here we used steady-state and pulse-labeling techniques to follow Notch receptors in sensory organ precursor cells in Drosophila. We find that the endosomal adaptor protein Numb regulates levels of Notch receptor trafficking to Rab7-labeled late endosomes but not early endosomes. Using an assay we developed that labels different pools of Notch receptors as they move through the endocytic system, we show that Numb specifically suppresses a recycled Notch receptor subpopulation and that excess Notch signaling in numb mutants requires the recycling endosome GTPase Rab11 activity. Our data therefore suggest that Numb controls the balance between Notch receptor recycling and receptor targeting to late endosomes to regulate signaling output after asymmetric cell division in Drosophila neural progenitors.     

Id4 regulates neural progenitor proliferation and differentiation in vivo

The mechanisms that determine whether a precursor cell re-enters the cell cycle or exits and differentiates are crucial in determining the types and numbers of cells that constitute a particular organ. Here, we report that Id4 is required for normal brain size, and regulates lateral expansion of the proliferative zone in the developing cortex and hippocampus. In its absence, proliferation of stem cells in the ventricular zone (VZ) is compromised. In early cortical progenitors, Id4 is required for the normal G1-S transition. By contrast, at later ages, ectopically positioned proliferating cells are found in the mantle zone of the Id4-/- cortex. These observations, together with evidence for the premature differentiation of early cortical stem cells, indicate that Id4 has a unique and complex function in regulating neural stem cell proliferation and differentiation.