Modulation of Oligodendrocyte Development by Voltage-Operated Calcium Channels

项目摘要/摘要 越来越多的证据表明，Cav1.2电压门控钙通道参与调节树突棘 神经细胞的形态和突触后的稳定性。Cav1.2通道形成信令复合体 突触后树突和树突棘，并与几种突触蛋白在功能上相互作用。我们有 最近证实，这些钙通道的活性对于充分的迁移、增殖至关重要 少突胶质前体细胞(OPC)的成熟。此外，我们的初步数据表明， Cav1.2活性与OPC中突触蛋白的表达有关，对正常 OPC与神经元的相互作用。因此，我们假设Cav1.2通道在突触中起作用 神经元之间的交流也调节神经元和OPC之间的突触信号。在这项研究中 计划中，我们将使用成像和电生理技术来研究Cav1.2通道如何调制 OPC和神经元之间的突触连接的形成。我们将确定是否增加Cav1.2 活动足以刺激OPC突触连接，我们将研究这些通道的活动如何 调节与OPC发育相关的基因的表达。提出了三个具体目标：在 第一个目标，我们将使用伪足亚细胞分离系统结合蛋白质组学和 RNA-Seq研究Cav1.2通道的活性如何影响OPC相关基因的表达 突触连接。然后，我们将通过电生理学和钙成像检查突触连接 其中Cav1.2通道和特定突触蛋白将被敲除的皮质OPC。在第二个目标中， 我们将使用一个小鼠模型，在该模型中，过度活跃的Cav1.2通道将在不同出生后的OPC中表达 时间点。这些OPC的发育和突触连接将通过结合以下几个方面进行研究 电生理学和RNA序列等技术。最后，我们建议使用化学遗传技术来 在大脑发育过程中影响OPC的电学特性，从而影响OPC的突触通讯。 通过Cre介导的重组，我们将在OPC中表达两种G蛋白偶联受体hM3Dq和hM4Di。 我们将评估质膜超兴奋性(HM3Dq)和超极化(HM4Di)如何改变 OPC和神经元之间突触的建立以及这些电变化如何影响OPC和神经元 OPC在出生后和成人大脑中的发育。解码OPC如何集成和 突触过程的输入对于理解大脑发育和提高大脑发育水平具有重要意义 受损白质的再髓鞘形成。我们假设Cav1.2通道是OPC的中心组件- 神经元突触是介导活性依赖的髓鞘形成的主要离子通道。

PROJECT SUMMARY/ABSTRACT Accumulating evidence implicate Cav1.2 voltage-gated Ca++ channels in regulating dendritic spine morphology and thereby postsynaptic stability in neurons. Cav1.2 channels form signaling complexes in postsynaptic dendrites and dendritic spines, and functionally interact with several synaptic proteins. We have recently established that the activity of these Ca++ channels is crucial for the adequate migration, proliferation and maturation of oligodendrocyte progenitor cells (OPCs). Furthermore, our preliminary data suggest that Cav1.2 activity is associated with the expression of synaptic proteins in OPCs and is essential for the normal interaction of OPCs with neurons. Thus, we hypothesize that Cav1.2 channels that function in synaptic communication between neurons also mediate synaptic signaling between neurons and OPCs. In this research plan, we will employ imaging and electrophysiological techniques to study how Cav1.2 channels modulate the formation of synaptic connections between OPCs and neurons. We will determine whether increase Cav1.2 activity is sufficient to stimulate OPC synaptic connectivity and we will study how the activity of these channels modulates the expression of genes associated with OPC development. Three specific Aims are proposed: in the first Aim, we will employ the pseudopod subcellular fractionation system in combination with proteomics and RNA-Seq to investigate how the activity of Cav1.2 channels affect the expression of genes associated with OPC synaptic connections. Then, we will examine by electrophysiology and Ca++ imaging the synaptic connectivity of cortical OPCs in which Cav1.2 channels and specific synaptic proteins will be knock-down. In the second Aim, we will use a mouse model in which overactive Cav1.2 channels will be expressed in OPC at different postnatal time-points. The development and synaptic connectivity of these OPCs will be studied by a combination of techniques such as electrophysiology and RNA-Seq. Finally, we propose to use chemo-genetic technologies to influence the electrical properties of OPCs and thus OPC synaptic communications during brain development. Via Cre-mediated recombination we will express two G-protein-coupled receptors in OPCs, hM3Dq and hM4Di. We will evaluate how plasma membrane hyperexcitability (hM3Dq) and hyperpolarization (hM4Di) modify the establishment of synapses between OPCs and neurons and how these electrical changes affect the development of OPCs in the postnatal as well as in the adult brain. Decoding how OPCs can integrate and process synaptic input is of fundamental importance for understanding brain development and for improving remyelination of damaged white matter. We hypothesize that Cav1.2 channels are central components of OPC- neuronal synapses and are the principal ion channels mediating activity-dependent myelination.