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: 8660718
• 负责人: Pablo Martin Paez
• 金额: $ 34.43万
• 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 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8660718
• 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; Signal Transduction; Site; Specificity; Staging; Stem cells; System; Tamoxifen; Testing; Transgenic Mice; Viral Vector; Work; cell motility; in vivo; knock-down; migration; mutant; myelination; 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）：已经清楚的是，电压操纵的Ca++通道（VOCC）的表达在少突胶质细胞谱系中受到高度调节，并且对于适当的OPC发育至关重要。了解电压依赖性Ca++内流的机制是重要的，因为细胞内Ca++的变化是许多细胞活动的中心。 例如，在少突胶质细胞祖细胞（OPC）中，电压依赖性Ca++内流在多种重要机制如突起延伸和细胞迁移中起关键作用（佩兹et al.，2009 a; B）。尽管有这些相关的发现，但人们对VOCC在OPC分化和髓鞘形成中的作用几乎一无所知。我们将测试电压门控Ca++进入促进出生后大脑中OPC成熟和髓鞘形成的假设，并确定少突胶质细胞VOCCs是否在髓鞘修复模型中发挥关键作用。本研究的主要目的有三：1）研究VOCCs对少突胶质细胞体外发育的影响。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Ca++摄取集中参与触发少突胶质细胞成熟。我们建议在体外敲低VOCC在少突胶质细胞中的表达，并测量细胞死亡，增殖和OPC分化。我们还将测试这些Ca++通道是否在体外共培养的髓鞘形成的第一步中促进轴神经胶质细胞信号传导 OPCs和皮层神经元的系统。2)通过特异性删除OPC中的L型VOCC同种型，测试电压门控Ca++进入是否促进体内OPC成熟和髓鞘形成。将表达L型VOCC的siRNA的病毒载体注射到新生幼崽的胼胝体和脑室下区中，以分析VOCC缺陷型OPC的体内迁移和髓鞘形成能力。此外，将通过将floxed突变CaV1.2小鼠与NG 2CreER ™转基因小鼠杂交来产生OPC中L型VOCC的条件性敲除小鼠，所述转基因小鼠在小鼠NG 2启动子的控制下表达他莫昔芬诱导型Cre重组酶。在杂交小鼠的新生幼崽中注射他莫昔芬将导致L型VOCC同种型CaV1.2在出生后特异性地在NG 2阳性OPC中缺失。3)检查VOCC消融术对髓鞘丢失和恢复的影响。我们的初步研究结果表明，在急性脱髓鞘的成年小鼠脑中，VOCCs作为OPC发展的潜在调节剂的作用。使用Cre-lox系统沉默Ca++通道表达，特别是在OPC，我们将测试电压依赖性Ca++进入促进OPC的生存和成熟的髓鞘再生成人大脑。为此，我们将使用脱髓鞘的cuprizone模型，该模型已被证明是分析成人脑中髓鞘丢失和髓鞘再生的有用工具。这些研究的成功完成将确定VOCC控制OPC发育和髓鞘形成的机制，以及这些Ca++通道在髓鞘病理学中的作用。这些发现可能会导致新的方法来干预髓鞘丢失或受损的神经退行性疾病。