Modulation of oligodendrocyte development by voltage-operated calcium channels.

描述(由申请人提供)：电压操纵型钙通道(VOCC)的表达在少突胶质细胞系中受到高度调控，对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很重要，因为细胞内钙离子的变化是许多细胞活动的中心。例如，在少突胶质前体细胞(OPC)中，电压依赖的钙内流在多种重要机制中发挥关键作用，如突起延伸和细胞迁移(Paez等，2009a；b)。尽管有这些相关的发现，但对于VOCC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OPC成熟和出生后脑内髓鞘形成的假设，并将确定少突胶质VOCC是否在髓鞘修复模型中发挥关键作用。我们提出了三个具体目标：1)检测VOCCs在体外对少突胶质细胞发育的作用。利用药理学工具和VOCC特异性siRNA，我们将测试VOCC是否通过电压门控钙摄取集中参与触发少突胶质细胞成熟。我们建议在体外下调VOCCs在少突胶质细胞中的表达，并检测细胞死亡、增殖和OPC分化。我们还将在体外共培养中测试这些钙离子通道是否有助于轴突胶质细胞在髓鞘形成的第一步中的信号传递。 OPC系统和皮质神经元。2)通过特异性缺失OPC中的L类VOCC异构体，检测电压门控钙离子内流是否促进OPC的成熟和髓鞘形成。将表达L类VOCC小干扰RNA的病毒载体注射到新生幼鼠的胼胝体和脑室下区，以分析VOCC缺陷OPC的体内迁移和髓鞘形成能力。此外，在小鼠NG2启动子的控制下，将突变的CaV1.2小鼠与表达他莫昔芬诱导的Cre重组酶的NG2CreERTM转基因小鼠杂交，将产生OPC中L型VOCC的条件基因敲除小鼠。杂交小鼠新生仔鼠注射三苯氧胺后，L VOCC亚型CaV1.2基因在NG2阳性OPC中生后特异性缺失。3)观察VOCC消融对髓鞘丢失和恢复的影响。我们的初步发现表明，在急性脱髓鞘的成年小鼠脑中，VOCCs可能是OPC发育的潜在调节器。使用Cre-lox系统沉默OPC中特异的钙通道表达，我们将测试电压依赖的Ca++进入是否促进OPC在重新髓鞘的成人脑中的存活和成熟。为此，我们将使用脱髓鞘的铜试剂模型，该模型已被证明是分析成人大脑中髓鞘丢失和再髓鞘形成的有用工具。这些研究的成功完成将确定VOCC通过哪些机制控制OPC的发育和髓鞘形成，以及这些钙离子通道在髓鞘病理中的作用。这些发现可能导致干预髓鞘丢失或受损的神经退行性疾病的新方法。

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.