Modulation of oligodendrocyte development by voltage-operated calcium channels.

描述（由申请人提供）：很明显，电压操作的Ca++通道（vocc）的表达在少突胶质谱系中受到高度调节，对于OPC的正常发育至关重要。了解电压依赖性钙离子内流的机制非常重要，因为细胞内钙离子的变化对许多细胞活动至关重要。例如，在少突胶质祖细胞（OPCs）中，电压依赖性钙++内流在过程延伸和细胞迁移等多种重要机制中发挥关键作用（Paez等，2009年a； b）。尽管有这些相关发现，但对于VOCCs在OPC分化和髓鞘形成中的作用几乎一无所知。我们将验证电压门控钙离子进入促进出生后大脑OPC成熟和髓鞘形成的假设，并确定少突胶质VOCCs是否在髓鞘修复模型中发挥关键作用。本文提出了三个具体目标：1)研究VOCCs在体外少突胶质细胞发育中的作用。使用药理学工具和VOCC特异性sirna，我们将测试VOCC是否通过电压门控ca2 +摄取集中参与触发少突胶质细胞成熟。我们拟通过抑制vocc在少突胶质细胞中的体外表达，检测细胞的死亡、增殖和OPC的分化。我们还将在体外共培养中测试这些Ca++通道是否在髓磷脂形成的第一步促进轴胶质信号传导

DESCRIPTION (provided by applicant): It has become clear that expression of voltage-operated Ca++ channels (VOCCs) is highly regulated in the oligodendroglial lineage and is essential for proper OPC development. Understanding the mechanisms of the voltage-dependent Ca++ influx is important as changes in intracellular Ca++ are central to many cellular activities. For example, in oligodendrocyte progenitor cells (OPCs) voltage-dependent Ca++ influx plays a key role in multiple important mechanisms such as process extension and cell migration (Paez et al., 2009a; b). Despite these relevant findings, next to nothing is known about the role of VOCCs in OPC differentiation and myelination. We will test the hypothesis that voltage-gated Ca++ entry promotes OPC maturation and myelination in the postnatal brain and we will determine if oligodendroglial VOCCs play a key role in a model of myelin repair. Three specific aims are proposed: 1) To examine the role of VOCCs on oligodendrocyte development in vitro. Using pharmacological tools and VOCC specific siRNAs we will test if VOCCs are centrally involved in triggering oligodendrocyte maturation through voltage-gated Ca++ uptake. We propose to knock down the in vitro expression of VOCCs in oligodendrocytes and measure cell death, proliferation and OPC differentiation. We will also test whether these Ca++ channels facilitate axo-glial signaling during the first steps of myelin formation in an in vitro co-culture system of OPCs and cortical neurons. 2) Test if voltage-gated Ca++ entry promotes OPC maturation and myelination in vivo by specifically deleting the L-type VOCC isoform in OPCs. Viral vectors expressing siRNAs for L-type VOCCs will be injected into the corpus callosum and the subventricular zone of newborn pups to analyze the in vivo migration and myelination capabilities of VOCC deficient OPCs. Additionally, a conditional knockout mouse for L-type VOCC in OPCs will be generated by crossing the floxed mutant CaV1.2 mice with the NG2CreERTM transgenic mice which express a tamoxifen-inducible Cre recombinase under the control of the mouse NG2 promoter. Injecting tamoxifen in newborn pups of the crossbred mice will result in the L-type VOCC isoform CaV1.2 being postnatally deleted specifically in NG2 positive OPCs. 3) To examine the effects of VOCC ablation in myelin loss and recovery. Our preliminary findings indicate a role for VOCCs as a potential modulator of OPC development in adult mouse brain in acute demyelination. Using the Cre-lox system to silence Ca++ channel expression specifically in OPCs, we will test if voltage-dependent Ca++ entry promotes OPC survival and maturation in the remyelinating adult brain. For that purpose, we will use the cuprizone model of demyelination which has proven to be a useful tool for the analysis of myelin loss and remyelination in the adult brain. Successful completion of these studies will define by which mechanisms VOCCs control OPC development and myelination, and the role of these Ca++ channels in myelin pathology. These findings could lead to novel approaches to intervene in neurodegenerative diseases in which myelin is lost or damaged.