Modulation of oligodendrocyte development by voltage-operated calcium channels.

通过电压控制的钙通道调节少突胶质细胞的发育。

• 批准号: 8846687
• 负责人: Pablo Martin Paez
• 金额: $ 41.13万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8846687
• 关键词: Ablation; Acute; Adult; Affect; Brain; CSPG4 gene; Calcium Channel; Cell Death; Cell Death Process; Cell Differentiation process; Cell Maturation; Cell Proliferation; Cell Survival; Cell membrane; Cells; Coculture Techniques; Corpus Callosum; Crossbreeding; Cuprizone; Data; Degenerative Disorder; Demyelinations; Development; Exons; Genes; Goals; In Vitro; Ion Channel; Knockout Mice; Lead; Measures; Mediating; Modeling; Molecular; Mus; Myelin; Nerve Tissue; Neuraxis; Neurodegenerative Disorders; Neurons; Newborn Infant; Oligodendroglia; Paper; Pathology; Pharmacological Treatment; Phase; Play; Process; Protein Isoforms; Publishing; Recovery; Role; Series; Site; Specificity; Staging; Stem cells; System; Tamoxifen; Testing; Transgenic Mice; Viral Vector; Work; cell motility; in vivo; knock-down; migration; mutant; myelination; neurotransmission; novel; novel strategies; postnatal; promoter; public health relevance; pup; recombinase; remyelination; repaired; research study; small hairpin RNA; subventricular zone; tool; uptake; voltage

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.

描述（由申请人提供）：很明显，电压操作的Ca ++通道（VOCC）的表达在寡头谱系中受到了高度调节，对于正确的OPC开发至关重要。了解电压依赖性Ca ++流入的机制很重要，因为细胞内Ca ++的变化对于许多细胞活性至关重要。 例如，在少突胶质细胞祖细胞（OPC）中，电压依赖性Ca ++涌入在多种重要机制（例如过程扩展和细胞迁移）中起关键作用（Paez等，2009a; b）。尽管有这些相关的发现，但几乎一无所知。我们将检验以下假设：电压门控的Ca ++条目促进了产后大脑中的OPC成熟和髓鞘化，我们将确定寡头胶质性VOCC是否在髓磷脂修复模型中起关键作用。提出了三个具体目标：1）检查VOCC在体外少突胶质细胞发育中的作用。使用药理学工具和VOCC特定siRNA，我们将测试VOCC是否在中央参与通过电压门控的Ca ++摄取来触发少突胶质细胞的成熟。我们建议击倒少突胶质细胞中VOCC的体外表达，并测量细胞死亡，增殖和OPC分化。我们还将测试这些CA ++通道是否促进在体外共培养中髓磷脂形成的第一步中的Axo-Glial信号传导 OPC和皮质神经元系统。 2）测试是否通过特异性删除OPC中的L型VOCC同工型来促进OPC成熟和体内的髓鞘化。表达L型VOCC的siRNA的病毒矢量将被注入call体和新生幼崽的室室内区域，以分析VIVO缺陷OPC的体内迁移和髓鞘化功能。此外，将通过将Floxed突变体CAV1.2小鼠与NG2CREERTM转基因小鼠跨越Floxed突变体CAV1.2小鼠的条件基因敲除小鼠，该小鼠在小鼠NG2启动子的控制下表达了他莫昔芬诱导的CRE重组酶。在越野小鼠的新生幼崽中注射他莫昔芬将导致L型VOCC同工型CAV1.2在NG2阳性OPC中专门删除后期删除。 3）检查VOCC消融对髓磷脂损失和恢复的影响。我们的初步发现表明，在急性脱髓鞘中，VOCC是成年小鼠脑中OPC发育的潜在调节剂的作用。使用CRE-LOX系统在OPC中专门沉默Ca ++通道表达，我们将测试电压依赖性Ca ++进入是否促进了Remyeleelialing的成年大脑中的OPC存活和成熟。为此，我们将使用脱髓鞘模型，该模型已被证明是分析成人大脑中髓磷脂损失和延迟性的有用工具。这些研究的成功完成将定义VOCC控制OPC发育和髓鞘的机制，以及这些CA ++通道在髓磷脂病理学中的作用。这些发现可能导致新的方法干预神经退行性疾病，其中髓磷脂丢失或损坏。