CNTF Receptors: Neuromuscular Protection/Repair In Vivo

描述（由申请人提供）：多项证据表明内源性睫状神经营养因子（CNTF）受体信号传导可促进神经肌肉保护和修复。因此，适当的CNTF受体信号转导的靶向可以通过适当地增强这些自然进化的机制来选择性地抵消神经肌肉疾病的破坏性影响。不幸的是，目前对成年人内源性CNTF受体功能的了解非常有限，因为体内受体的阻断（通过破坏关键的CNTF受体α [CNTFRa]基因）导致围产期死亡。为了克服这个问题，我们将使用Cre/lox技术来选择性地破坏CNTFRa基因。具体目标1将检查CNTF受体信号传导对成年运动神经元的存活、保护和表型维持的贡献。CNTFR a基因将在“floxed”CNTFR a小鼠的面部运动神经元中被破坏，其中：1）用指导Cre重组酶（Cre）表达的腺相关病毒立体定位注射面部核，和2）能够暂时控制Cre活性诱导的基因构建体。初步数据表明CNTFRa在成年运动神经元存活中的重要体内作用。我们将用报告基因、免疫组织化学和体视学细胞计数来定量表征这种功能，并确定是否需要损伤来激活这种神经保护系统（如初步数据所示），如果需要，则需要何种形式的损伤。此外，报告基因和免疫组织化学将被用来确定是否CNTF受体信号保持运动神经元胆碱能表型在体内。最后，将通过原位杂交和免疫组织化学鉴定可能参与神经保护的信号蛋白。具体目标2将定义骨骼肌CNTFRa在神经肌肉保护和修复中的作用。来自具有骨骼肌特异性Cre表达的floxed CNTFRa小鼠的初步数据显示，肌肉CNTFRa是外周神经损伤后正常运动恢复所需的。我们将用“足迹”分析来表征这种功能缺陷，并通过定量来确定潜在的细胞机制：1）神经肌肉接头形成，2）运动神经元存活和胆碱能表型，以及3）肌肉收缩性。此外，基于STAT 3的免疫组织化学将用于定位神经损伤后肌肉-CNTFRa依赖性信号传导的细胞位点。此外，神经损伤后的肌肉和脊髓腹侧灰质的微阵列分析将用于识别可能参与关键CNTF受体信号传导的新候选基因。用通俗的语言说：我们将确定小鼠中天然CNTF受体信号传导如何保护和修复成人神经肌肉系统。由此产生的知识应该有助于设计治疗方法，选择性地增强适当的CNTF受体信号传导，从而有效地治疗神经肌肉疾病，同时避免副作用。

DESCRIPTION (provided by applicant): Several lines of evidence suggest that endogenous ciliary neurotrophic factor (CNTF) receptor signaling may promote neuromuscular protection and repair. Thus, proper targeting of CNTF receptor signaling may selectively counteract the devastating effects of neuromuscular disorders by appropriately enhancing these naturally evolved mechanisms. Unfortunately, current understanding of endogenous CNTF receptor function in the adult is extremely limited because blockage of the receptor in vivo (through disruption of the critical CNTF receptor a [CNTFRa] gene) leads to perinatal death. To overcome this problem, we will use Cre/lox techniques to selectively disrupt the CNTFRa gene. Specific Aim 1 will examine the contribution of CNTF receptor signaling to the survival, protection and phenotype maintenance of adult motor neurons. The CNTFRa gene will be disrupted in facial motor neurons of "floxed" CNTFRa mice with: 1) stereotaxic injection of the facial nucleus with an adeno-associated virus that directs Cre recombinase (Cre) expression and 2) a gene construct enabling temporally controlled induction of Cre activity. Preliminary data indicate an essential in vivo role for CNTFRa in adult motor neuron survival. We will quantitatively characterize this function with reporter genes, immunohistochemistry and stereological cell counting and also determine whether insult is required to activate this neuroprotective system (as preliminary data suggest), and if so, what forms of insult. In addition, a reporter gene and immunohistochemistry will be used to determine whether CNTF receptor signaling maintains motor neuron cholinergic phenotype in vivo. Finally, signaling proteins potentially involved in the neuroprotection will be identified through in situ hybridization and immunohistochemistry. Specific Aim 2 will define the role of skeletal muscle CNTFRa in neuromuscular protection and repair. Preliminary data from floxed CNTFRa mice with skeletal muscle specific Cre expression reveal that muscle CNTFRa is required for normal motor recovery following peripheral nerve lesion. We will characterize this functional deficit with "footprint" analysis and identify the underlying cellular mechanisms by quantifying: 1) neuromuscular junction formation, 2) motor neuron survival and cholinergic phenotype, and 3) muscle contractility. In addition, STAT3-based immunohistochemistry will be used to localize cellular sites of muscle-CNTFRa-dependent signaling after nerve lesion. Moreover, microarray analysis of muscle and spinal cord ventral grey matter following nerve lesion will be used to identify novel candidate genes potentially involved in the critical CNTF receptor signaling. Relevance in lay language: We will determine how natural CNTF receptor signaling in mice protects and repairs the adult neuromuscular system. The resulting knowledge should facilitate the design of therapeutics which selectively enhance the appropriate CNTF receptor signaling and thereby effectively treat neuromuscular disease while avoiding side effects.