Nuclear function of L1-type CAMs in the drosophila nervous system

 描述(由申请人提供)：L1型细胞黏附分子(CAM)是神经系统正常发育所必需的。我们先前已经证明，唯一的无脊椎动物L1型CAM神经胶质细胞(NRG)的跨突触信号调节突触的生长和稳定性，其在突触组织细胞骨架中的功能从苍蝇到人类都是保守的。初步的活体成像数据显示，在成人中，NRG以LissBray1(LIS1)依赖的方式从突触逆行运输到胞体。这表明在成人中，L1型CaM具有一种未知的功能，这与它在质膜上众所周知的发育作用截然不同。逆行运输的L1型CaM可能在胞体内降解或再循环，成为“信号内体”的一部分，或者是逆行信号本身，转移到细胞核。最近，细胞质中28 kDa和30 kDa的L1CAM片段以及一个70 kDa的跨膜区片段被发现移位到细胞核中。我们有证据表明，除了片段外，全长NRG还易位到果蝇神经系统的细胞核，并且NRG片段可能带有不同于全长形式的翻译后修饰。我们的假设S认为，从突触运输到胞体的一些NRG将转移到细胞核。为了测试这一点，我们将确定与野生型背景相比，Lis1突变体中NRG的核进口是否减少，以及全长NRG形式及其片段是否进行了不同的翻译后修饰。除了核转位，我们在目标1中提出了确定L1型CAM逆行运输的机制。我们将使用实时成像来确定发生NRG轴突逆行运输的内体间隔的类型以及NRG逆行运输所需的细胞内模体。L1CAM的胞内区(ICD)在非神经细胞系中的重组表达改变了参与迁移、细胞周期控制和DNA损伤检查点反应的基因的表达，体外实验表明，核L1CAM可能在迁移、突起生长和细胞保护中发挥作用，以抵御疾病或环境影响引起的物理损伤或基因组和氧化应激。目标2的主要目标是确定核L1型凸轮是否在体内也具有功能作用。为此，我们将表征一个缺乏跨膜近端核定位序列的突变体的表型。此外，我们将在果蝇神经系统中转基因表达模仿果蝇核全长L1型蛋白及其片段的L1型CAM蛋白，通过在单细胞和生物水平上分析它们在体内诱导的表型来确定它们是否具有不同的功能。综上所述，中枢神经系统核L1-型细胞间黏附分子信号机制和功能的体内表征将影响广泛的研究领域，如中风时的神经保护、脊髓再生、阿尔茨海默病和癌症。

 DESCRIPTION (provided by applicant): L1-type cell adhesion molecules (CAM) are essential for proper nervous system development. We previously demonstrated that transsynaptic signaling of the sole invertebrate L1-type CAM neuroglian (Nrg) regulates synapse growth and stability and that its function in organizing the cytoskeleton at the synapse is conserved from flies to humans. Preliminary live imaging data reveals that in the adult Nrg is retrogradely transported from the synapse to the soma in a Lissencephaly 1 (Lis1)-dependent manner. This suggests that in the adult L1-type CAMs have a yet uncharacterized function that is distinct from its well-known developmental role at the plasma membrane. Retrogradely transported L1-type CAMs may be degraded or recycled in the soma, be part of a "signaling endosome", or be retrograde signals themselves, which translocate to the nucleus. Recently, cytosolic 28kDa and 30KDa L1CAM fragments as well as a 70kDa transmembrane domain- containing fragment were shown to translocate to the nucleus. We have evidence that in addition to fragments, full-length Nrg translocates to the nuclei of Drosophila nervous systems and that Nrg fragments are likely to carry posttranslational modifications distinct from the full-length form. Our hypothesis s that some of the Nrg that is transported from the synapse to the soma will translocate to the nucleus. To test this, we will determine if the nuclear import of Nrg is reduced in Lis1 mutants when compared to wild type background and if the full- length Nrg form and its fragments carry different posttranslational modifications. In addition to nuclear translocation, we propose in aim 1 to determine mechanisms of L1-type CAM retrograde transport. We will use live imaging to determine the type of endosomal compartments in which axonal retrograde transport of Nrg occurs and the intracellular motifs that are required for Nrg retrograde transport. Recombinant expression of the intracellular domain (ICD) of L1CAM in non-neuronal cell lines altered the expression of genes involved in migration, cell cycle control and DNA damage checkpoint responses and in vitro experiments suggest that nuclear L1CAM may have a role in migration, neurite outgrowth and cellular protection against physical damage or genomic and oxidative stress caused by diseases or environmental influences. The main goal of aim 2 is to determine if nuclear L1-type CAMs have a functional role in vivo as well. For this, we will characterize the phenotypes of a mutant that lacks the transmembrane proximal nuclear localization sequence. In addition, we will transgenically express L1-type CAM proteins that mimic nuclear full- length L1-type protein and its fragments in the nervous system of Drosophila to determine if they have distinct functions by analyzing their induced phenotypes in vivo at the single cell and the organismal levels. In summary, the in vivo characterization of the mechanisms and functions of nuclear L1-type CAM signaling in the central nervous system will impact a broad range of research areas such as neuroprotection during stroke, spinal cord regeneration, Alzheimer's disease and cancer.