Functional analysis of erbB2 signaling in myelin-forming glial cells

描述（由申请人提供）：本研究的长期目标是确定参与周围神经系统（PNS）脱髓鞘和髓鞘再生的分子机制。脱髓鞘与全部或部分神经元功能的丧失有关，因为髓鞘形成神经胶质细胞和轴突之间存在密切联系。因此，预防脱髓鞘或促进髓鞘再生是PNS和CNS的神经退行性疾病的重要治疗目标。我们和其他人最近已经表明受体酪氨酸激酶erbB 2的功能与触发PNS脱髓鞘有关。ErbB 2功能也参与促进PNS髓鞘形成。erbB 2在PNS中发挥两种相反作用的机制尚不清楚。了解受体信号传导机制对预防脱髓鞘性神经退行性疾病和再生药物具有重要意义。本研究的主要目的是确定erbB 2在损伤诱导的PNS脱髓鞘中的作用。提出了三个具体目标：1）确定erbB 2促进PNS脱髓鞘的机制，2）确定erbB 2活化是否足以诱导PNS脱髓鞘，3）确定与erbB 2脱髓鞘功能相关的信号转导机制。该研究计划利用了一种新的体外区室化雪旺细胞-神经元共培养系统，该系统重现了体内雪旺细胞神经损伤反应。该系统作为一个强大的工具，研究信号转导使用药理学和遗传干预。为了研究erbB 2的功能（目的1），我们将使用腺病毒递送系统在PNS脱髓鞘的不同阶段在成年髓鞘化雪旺细胞中表达显性阴性erbB 2突变体，这将使我们能够在体内和体外实现有条件的erbB 2抑制。对于目标2中的erbB 2功能获得性研究，我们将使用创新的受调控erbB 2同源和异源二聚化系统。该系统使我们能够产生异位erbB 2信号髓鞘雪旺细胞内，独立的内源性受体和配体。对于目标3中提出的信号传导功能，我们将使用生物化学和成像方法来定义与受体脱髓鞘功能相关的erbB 2信号传导机制。公共卫生相关性脱髓鞘是许多PNS神经退行性疾病的常见病理特征，包括Charcot-Marie-Tooth综合征、Guillian-Barre综合征、继发于糖尿病和癌症化疗的神经病变、感染性神经病和与神经胶质损伤相关的运动缺陷。脱髓鞘还与全部或部分神经元功能的丧失有关，因为髓鞘形成神经胶质细胞和轴突之间存在密切联系。因此，预防脱髓鞘是PNS和CNS的神经退行性疾病的重要治疗目标。最近的研究表明，erbB 2激活可能在介导PNS脱髓鞘中发挥作用。因此，受体或相关细胞质效应物的异常激活可能是广泛的脱髓鞘疾病的基础。了解启动脱髓鞘的信号机制可能对开发阻断erbB 2激活的疗法或在神经退行性疾病进一步进展之前检测早期脱髓鞘的诊断测试具有意义。

DESCRIPTION (provided by applicant): The long-term objective of this research is to define molecular mechanisms that are involved in demyelination and re-myelination of the peripheral nervous system (PNS). Demyelination is linked to the loss of all or part of neuronal functions because of the intimate connection between myelinating glial cells and axons. Therefore, the prevention of demyelination or promoting re-myelination is an important therapeutic objective for neurodegenerative diseases of the PNS and the CNS. We and others have shown recently that a function of receptor tyrosine kinase, erbB2, is associated with triggering PNS demyelination. ErbB2 function is also involved in promoting PNS myelination. The mechanisms by which erbB2 elicits two opposing roles in the PNS is unknown. Understanding the receptor signaling mechanisms has important implications for prevention of demyelinating neurodegenerative diseases and regenerative medicines. The broad goal of the present study is to define erbB2 function in initiating injury-induced PNS demyelination. Three specific aims are proposed: 1) determine the mechanism by which erbB2 promotes PNS demyelination, 2) determine whether erbB2 activation is sufficient to induce PNS demyelination and 3) determine the mechanism of signal transduction that is associated with the demyelinating function of erbB2. The study plan exploits a new in vitro compartmentalized Schwann cell-neuron co-culture system that recapitulates Schwann cell nerve injury responses in vivo. The system serves as a powerful tool for studying signal transduction using both pharmacological and genetic intervention. To investigate erbB2 function (Aim 1), we will use an adenoviral delivery system to express dominant-negative erbB2 mutant in adult myelinating Schwann cells, during different stages of PNS demyelination, which will enable us to achieve a conditional erbB2 inhibition both in vivo and in vitro. For the erbB2 gain-of-function study in Aim 2, we will use an innovative regulated erbB2 homo- and hetero-dimerization system. This system allows us to generate ectopic erbB2 signaling within myelinating Schwann cells, independent of the endogenous receptor and the ligand. For the signaling function raised in Aim 3, we will use both biochemical and imaging approaches to define erbB2-signaling mechanism that is associated with the demyelinating function of the receptor. PUBLIC HEALTH RELEVANCE Demyelination is a common pathologic feature in many PNS neurodegenerative diseases including Charcot-Marie-Tooth syndrome, Guillian-Barre syndrome, neuropathies secondary to diabetes and cancer chemotherapy, infectious neuropathy and motor deficits related to glial injury. Demyelination is also linked to the loss of all or part of neuronal functions because of the intimate connection between myelinating glial cells and the axons. Therefore, the prevention of demyelination is an important therapeutic objective for neurodegenerative diseases of the PNS and the CNS. Recent studies suggest that erbB2 activation might play a role in mediating PNS demyelination. Therefore, aberrant activation of the receptor or the associated cytoplasmic effectors may underlie a wide range of demyelinating disorders. Understanding the signal mechanism that initiates demyelination may have implications for the development of therapies that block erbB2 activation or diagnostic tests for detecting early demyelination before further progression of neurodegeneration.