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Induction of differentiation of neural cells by remodeling of ganglioside synthesis and expression

通过重塑神经节苷脂合成和表达诱导神经细胞分化

基本信息

批准号：
07670159
负责人：
KOJIMA Naoya
金额：
$ 0.38万
依托单位：
The Institute of Physical and Chemical Research (RIKEN)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

We showed previously that introduce of the cDNA encoding GD3 synthase into mouse neuroblastoma Neuro2a cells led to convert surface major gangliosides of the cells from GM1 and GD1a to GD3 and GQ1b, and subsequently cell were differentiated with neurite sprouting.To gain a better understanding of consequences of GD3 synthase expressing in Neuro2a cells and explain the possible mechanism responsible for the Neuro2a morphological change, we first search the regulatory expression systems for sialyltransferase. We found that the recent developed tetracycline regulated system was suitable to control expression of sialyltransferase cDNA.Then, we applied this system to Neuro2a cells and GD3 synthase cDNA.Under this system, Neuro2a was differentiated into cholinergic-like neuronal cells with neurite sprouting 20 days after induction of GD3 synthase, although GD3 synthase mRNA was expressed 4 h after induction and kept at a low level during the process. Recently, we have identified several gene … More s that were involved this differentiation process by differential display method. Now we are analyzing these new genes.To examine the importance of cell surface gangliosides for neural cell morphology and cellular functions, cDNA encoding alpha1,2-fucosyltransferase was introduced into Neuro2a cells. After transfection of the cDNA,the cells expressed fucosyl-GM1 as the major ganglioside. It is known that Neuro2a cells sprout a axon-like neurite under serum free condition. However, fucosyltransferase cDNA-transfected cells sprout several dendrite-like neurites under serum-free condition. This phenomenon was revered by removing of fucosyl-GM1 from cell surface.Our results strongly suggest that cell surface gangliosides play a role for neuronal cell behavior and differentiation. In addition, stable transfection of the glycosyltransferases into cells, particularly under regulated system, is a good approach for analyzing of the functions of gangliosides and subsequent signal transduction mechanisms in neuronal cells. Less

我们之前的研究表明，将编码GD3合成酶的cDNA引入小鼠神经母细胞瘤Neuro2a细胞，可将细胞表面的主要神经节苷脂从GM1和GD1a转化为GD3和GQ1b，随后细胞分化并出现神经突萌发。为了更好地了解GD3合成酶在Neuro2a细胞中表达的后果，并解释导致Neuro2a细胞形态变化的可能机制，我们首先搜索了唾液基转移酶的调控表达系统。我们发现最近建立的四环素调控系统适合控制唾液基转移酶cDNA的表达。然后，我们将该系统应用于Neuro2a细胞和GD3合成酶cDNA。在该体系下，虽然GD3合成酶mRNA在诱导后4 h表达并保持低水平，但在GD3合成酶诱导20天后，Neuro2a分化为胆碱能样神经元细胞，并出现神经突萌芽。近年来，我们通过差异显示方法鉴定出了参与这一分化过程的多个基因。现在我们正在分析这些新基因。为了研究细胞表面神经节苷对神经细胞形态和细胞功能的重要性，我们将编码alpha1,2- focusyltransferase的cDNA引入Neuro2a细胞。转染cDNA后，细胞表达focusyl-GM1为主要的神经节苷脂。已知在无血清条件下，Neuro2a细胞会萌发轴突样神经突。然而，转染了focusyltransferase cdna的细胞在无血清的条件下会长出几个树突样神经突。这种现象是通过从细胞表面去除focusyl-GM1来实现的。我们的研究结果强烈表明，细胞表面神经节苷在神经元细胞的行为和分化中起作用。此外，将糖基转移酶稳定转染到细胞中，特别是在调控系统下，是分析神经节苷脂功能和随后的神经细胞信号转导机制的良好方法。少