Biology & Pharmaclogy of the GDNF Family of Ligands

描述（由申请人提供）：在本项目的上一个资助期内， IRPG赠款提交与杰弗里米尔布兰特，我们报告了发现 神经营养因子neurturin、persephin和artemin，它们沿着 GDNF，构成GFL神经营养因子家族。我们和其他人也 发现了GPI锚定的neurturin和artemin共受体。许多 其他问题，我们已经解决了GFLs的信号转导， 脂筏和细胞质酪氨酸的关键作用的证据 激酶C-src介导GFL作用。在初步实验中，我们 在感觉和交感神经元中发现了一种新的，非GFL介导的， 受体激酶Ret的成熟依赖性磷酸化。令人惊奇的是， 这种依赖成熟的磷酸化，至少在交感神经元中， 似乎依赖于神经生长因子我们还观察到了 GFLs在离体基底前脑神经元上的表达及基底前脑神经元的萎缩性改变 在体内来自老龄neurturin-/-小鼠的前脑神经元。我们要表演 实验（目的1）了解生化机制，细胞 特异性和一般性的配体独立的，成熟依赖的 Ret的磷酸化。我们将在体外和体内进行实验（目标2）。 体内，以评估这种现象的潜在生物学意义。我们将 检查（目的3A）Ret相互作用的结构/生化基础 并使用src-/-小鼠的细胞来严格评估C-src 在介导GFL的行动，并确定是否神经元群体依赖于 在src-/-小鼠中显示神经元损失和/或萎缩（目的3B和3C）。我们 将完成实验（目标4），以进一步确定 GFL信号装置的相互作用，并确定 GPI锚定的辅助受体用于激活Ret的脂筏环境， 对GPL刺激的反应。我们将研究（目标5）GFLs在 基底前脑神经元在体外，检查其作为神经保护剂的潜力 并通过以下方法检查neurturin-/-小鼠年龄依赖性神经变性 解剖学、神经化学和行为学标准。最后，我们将继续 与米尔布兰特实验室合作， IRPG配套赠款，包括对青蒿素和 persephin敲除动物。

DESCRIPTION (provided by applicant): In the previous funding period of this IRPG grant submitted with that of Jeffrey Milbrandt, we reported the discovery of neurotrophic factors neurturin, persephin, and artemin, which along with GDNF, constitute the GFL family of neurotrophic factors. We, and others, also discovered GPI-anchored co-receptors for neurturin and artemin. Among many other issues, we have addressed signal transduction of GFLs and provided evidence for a critical role for lipid rafts and the cytoplasmic tyrosine kinase C-src in mediating GFL action. In preliminary experiments we have discovered in sensory and sympathetic neurons a novel, non-GFL-mediated, maturation-dependent phosphorylation of the receptor kinase Ret. Surprisingly, this maturation-dependent phosphorylation, at least in sympathetic neurons, appears reliant upon nerve growth factor. We have also observed robust effects of GFLs on basal forebrain neurons in vitro and atrophic changes in basal forebrain neurons from aged neurturin-/- mice in vivo. We shall perform experiments (Aim 1) to understand the biochemical mechanism, cellular specificity, and generality of the ligand-independent, maturation-dependent phosphorylation of Ret. We shall do experiments (Aim 2) both in vitro and in vivo to assess potential biological significance of this phenomenon. We shall examine (Aim 3A) the structural/biochemical basis of the interaction of Ret with C-src and use cells from src-/- mice to assess critically the role C-src in mediating GFL actions and determine whether neuronal populations dependent on GFLs show neuronal loss and/or atrophy in src-/- mice (Aims 3B and 3C). We shall complete experiments (Aim 4) to define further the role and mechanisms of the interaction of the GFL-signaling apparatus and to determine the nature of the lipid-raft environment used by GPI-anchored co-receptors to activate Ret in response to GPL stimulation. We shall study (Aim 5) the role of the GFLs in basal forebrain neurons in vitro, examine their potential as neuroprotectants in vivo, and examine neurturin-/- mice for age-dependent neurodegeneration by anatomical, neurochemical, and behavioral criteria. Lastly, we shall continue to collaborate with the Milbrandt lab in the execution of the Aims in the companion IRPG grant, including analysis of the artemin- and persephin-knock-out animals.