Beta 1 integrin-mediated effects of tenascin-R domains EGFL and FN6-8 on neural stem/progenitor cell proliferation and differentiation in vitro

Neural stem/progenitor cells (NSCs) have the capacity for self-renewal and differentiation into major classes of central nervous system cell types, such as neurons, astrocytes, and oligodendrocytes. The determination of fate of NSCs appears to be regulated by both intrinsic and extrinsic factors. Mounting evidence has shown that extracellular matrix molecules contribute to NSC proliferation and differentiation as extrinsic factors. Here we explore the effects of the epidermal growth factor-like (EGFL) and fibronectin type III homologous domains 6-8 (FN6-8) of the extracellular matrix molecule tenascin-R on NSC proliferation and differentiation. Our results show that domain FN6-8 inhibited NSC proliferation and promoted NSCs differentiation into astrocytes and less into oligodendrocytes or neurons. The EGFL domain did not affect NSC proliferation, but promoted NSC differentiation into neurons and reduced NSC differentiation into astrocytes and oligodendrocytes. Treatment of NSCs with beta 1 integrin function-blocking antibody resulted in attenuation of inhibition of the effect of FN6-8 on NSC proliferation. The influence of EGFL or FN6-8 on NSCs differentiation was inhibited by beta 1 integrin antibody application, implicating beta 1 integrin in proliferation and differentiation induced by EGFL and FN6-8 mediated triggering of NSCs.     

Interactions between Rho GTPases and Rho GDP dissociation inhibitor (Rho-GDI)

The Rho-GDP dissociation inhibitor (Rho-GDI) was used as bait in a two-hybrid screen of a human leucocyte cDNA library. Most of the isolated cDNAs encoded GTPases of the Rho subfamily: RhoA, B, C, Rac1, 2, CDC42 and RhoG. The newly discovered RhoH interacted very poorly with Rho-GDI. Another protein partner shared a homology with RhoA that points to Asp67(RhoA)-Arg68(RhoA)-Leu69(RhoA) as critical for interaction with Rho-GDI. A second screen with RhoA as bait led to the isolation of GDI only. In order to investigate the relative role of protein-protein and protein-lipid interactions between Rho GTPases and Rho-GDI, CAAX box mutants of RhoA were produced. They were found to interact with Rho-GDI as efficiently as wild type RhoA, indicating that protein-protein interactions alone lead to strong binding of the two proteins. The C-terminal polybasic region of RhoA was also shown to be a site of protein-protein interaction with Rho-GDI.     

磷脂酶D1信号对神经干细胞向神经分化的重要影响

磷脂酶D1(PLD1)在细胞生长、存活、分化、膜转运和细胞骨架组织等多种功能的调控中发挥重要作用。近年来研究发现,PLD1在神经干细胞（NSCs）向神经元的分化中也起关键作用。PLD1参与多种信号通路如Rho家族GTP酶和Ca²⁺信号通路的调节,影响轴突生长、突触发育及其可塑性。因此,PLD1作为神经系统中一种重要的信号分子引起了广泛的关注。本文综述了PLD1的结构、功能、作用机制及其在NSCs向神经分化中的调控作用,对深入研究NSCs的分化和神经元的再生有重要的指导意义。     

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.     

PKC mediates cyclic stretch-induced cardiac hypertrophy through Rho family GTPases and mitogen-activated protein kinases in cardiomyocytes

Signaling events, including Rho GTPases and protein kinase C (PKC), are involved in cardiac hypertrophy. However, the mechanisms by which these pathways cooperate during the hypertrophic process remain unclear. Using an in vitro cyclic stretch model with neonatal rat cardiomyocytes, we demonstrated that stretch-induced activation of RhoA, Rac1/Cdc42, and phosphorylation of Rho-guanine nucleotide dissociation inhibitor (GDI) were prevented by inhibition or depletion of PKC, using chelerythrine and phorbol 12-myristate 13-acetate, indicating that phorbol ester-sensitive PKC isozymes may be upstream regulators of Rho GTPases. Using adenoviral-mediated gene transfer of wild-type (WT) and dominant-negative (DN) mutants of PKCalpha and delta, we found that stretch-induced activation of Rho GTPases and phosphorylation of Rho-GDI were mainly regulated by PKCalpha. PKCdelta was involved in regulation of the activation of Rac1. Stretch-induced increases in [(3)H]-leucine incorporation, myofibrillar reorganization and cell size, were blocked by inhibition of Rho GTPases, or overexpression of DN PKCalpha and delta, suggesting that PKCalpha and delta are both required in stretch-induced hypertrophy, through Rho GTPases-mediated signaling pathways. The mechanism, whereby PKC and Rho GTPases regulate hypertrophy, was associated with mitogen-activated protein (MAP) kinases. Stretch-stimulated phosphorylation of MEK1/ERK1/2 and MKK4/JNK was inhibited by overexpression of DN PKCalpha and delta, and that of MKK3/p38 inhibited by DN PKCdelta. The phosphorylation of ERK and JNK induced by overexpression of WT PKCalpha, and the phosphorylation of p38 induced by WT PKCdelta, were regulated by Rho GTPases. This study represents the first evidence that PKCalpha and delta are important regulators in mediating activation of Rho GTPases and MAP kinases, in the cyclic stretch-induced hypertrophic process.     

Egr-1的诱骗性寡脱氧核苷酸抑制大鼠颈总动脉损伤后MMP-2的表达

目的观察局部转染早期生长反应因子-1(early growth response factor-1,Egr-1)的特异诱骗寡脱氧核苷酸(decoy oligodeoxynucleotides,decoy ODNs)对球囊损伤颈总动脉后基质金属蛋白酶-2(MMP-2)蛋白表达的影响及内膜增生的情况,初步探讨Egr-1,decoy ODNs抑制球囊损伤后内膜增生的机制。方法 96只健康雄性Wistar大鼠,随机分为4组,分别为假手术组、对照组、杂码组和诱骗组,每组24只。除假手术组外均应用2F球囊导管行颈总动脉球囊损伤术,术中采用转染试剂FuGENE6介导的Egr-1decoy ODNs转染至损伤后大鼠血管中,与假手术组、对照组、杂码组相比较。术后3、7、14、21d每组处死6只动物。应用HE染色和免疫组织化学方法观察大鼠颈总动脉球囊损伤后内膜增生情况和MMP-2蛋白的表达及转染Egr-1decoy ODNs后对它们的影响。结果 (1)、内膜损伤后3d内膜增厚不明显,7d内膜开始增厚,14、21d时内膜明显增厚。(2)、在假手术组近腔面中膜可见MMP-2有少量散在阳性表达;在对照组及杂码组动脉损伤后3d,在近腔面中膜,有少量阳性表达,与假手术组相比,阳性表达指数上升。7d时在新生内膜和靠近新生内膜处中膜表达明显,14d后表达逐渐下降。(3)转染decoy ODNs治疗后,在各个时间点内膜增厚程度减轻,MMP-2蛋白表达减少,与对照组比较差异有显著性(P<0.01)。结论血管球囊损伤后,内膜7d开始增生,14d、21d增生更明显,而MMP-2在7d时表达明显,之后逐渐下降,Egr-1decoy ODNs能抑制MMP-2的表达,从而减轻血管损伤后内膜的增生。     

Efficient dendritic cell priming of T lymphocytes depends on the extracellular matrix protein mindin

Rho guanosine triphosphatases (GTPases) regulate multiple aspects of dendritic cell (DC) function, but what regulates the expression of Rho GTPases in DCs is unknown. Here, we show that the extracellular matrix protein mindin regulates the expression of Rho GTPases in DCs. Mindin(-/-) mice displayed defective CD4+ T-cell priming and impaired humoral immune responses to T-dependent antigens. Mindin(-/-) DCs had reduced expression of Rac1/2 and impaired priming capacity owing to inefficient engagement with T lymphocytes. Ectopic Rac1 expression restored the priming capability of Mindin(-/-) DCs. Furthermore, we show that DC adhesion to mindin matrix was blocked by antibodies to alpha4, alpha5 and beta1 integrins. DCs lacking beta1 integrin had reduced adhesion to mindin matrix, decreased expression of Rac1/2 and impaired priming capacity. These results suggest that mindin-integrin interactions play a key role in regulating Rho GTPase expression in DCs and DC priming of T lymphocytes.     

miR-124 promotes proliferation and differentiation of neuronal stem cells through inactivating Notch pathway

Background

Neural stem cells (NSCs) are able to differentiate into neurons and astroglia. miRNAs have been demonstrated to be involved in NSC self-renewal, proliferation and differentiation. However, the exact role of miR-124 in the development of NSCs and its underlying mechanism remain to be explored.

Methods

Primary NSCs were isolated from embryos of Wistar rats. Immunocytochemistry was used to stain purified NSCs. miR-124, Delta-like 4 (DLL4), ki-67, Nestin, β-tubulin III, glial fibrillary acidic protein (GFAP), HES1, HEY2, and cyclin D1 (CCND1) expressions were detected by qRT-PCR and western blot. The interaction between miR-124 and DLL4 was confirmed by luciferase reporter assay. Cell proliferation was assessed by MTT assay.

Results

NSCs could self-proliferate and differentiate into neurons and astrocyte. miR-124 was up-regulated and DLL4 was down-regulated during NSC differentiation. DLL4 was identified as a target of miR-124 in NSCs. Ectopic expression of miR-124 or knockdown of DLL4 promoted the proliferation and the formation of NSCs to neurospheres. Moreover, miR-124 overexpression or DLL4 down-regulation improved β-tubulin III expression but decreased GFAP expression in NSCs. Furthermore, enforced expression of DLL4 partially reversed the effects of miR-124 on NSCs proliferation and differentiation. Elevated expression of miR-124 suppressed the expressions of HES1, HEY2, and CCND1 in NSCs, while these effects were attenuated following the enhancement of DLL4 expression.

Conclusion

miR-124 promoted proliferation and differentiation of NSCs through inactivating Notch pathway.

     

Circular RNA expression profiles during the differentiation of mouse neural stem cells

Background

Circular RNAs (circRNAs) have recently been found to be expressed in human brain tissue, and many lines ofevidence indicate that circRNAs play regulatory roles in neurodevelopment. Proliferation and differentiation of neural stem cells (NSCs) are critical parts during development of central nervous system (CNS).To date, there have been no reports ofcircRNA expression profiles during the differentiation of mouse NSCs. We hypothesizethat circRNAs mayregulate gene expression in the proliferation anddifferentiation of NSCs.

Results

In this study, we obtained NSCs from the wild-type C57BL/6 J mouse fetal cerebral cortex. We extracted total RNA from NSCs in different differentiation stagesand then performed RNA-seq. By analyzing the RNA-Seq data, we found 37circRNAs and 4182 mRNAs differentially expressedduringthe NSC differentiation. Gene Ontology (GO) enrichment analysis of thecognate linear genes of these circRNAsrevealed that some enriched GO terms were related to neural activity. Furthermore, we performed a co-expression network analysis of these differentially expressed circRNAs and mRNAs. The result suggested a stronger GO enrichmentin neural features for both the cognate linear genes of circRNAs and differentially expressed mRNAs.

Conclusion

We performed the first circRNA investigation during the differentiation of mouse NSCs. Wefound that12 circRNAs might have regulatory roles duringthe NSC differentiation, indicating that circRNAs might be modulated during NSC differentiation.Our network analysis suggested the possible complex circRNA-mRNA mechanisms during differentiation, and future experimental workis need to validate these possible mechanisms.