Isolation and Characterization of Synaptogenic Proteins

描述（由申请人提供）：本研究项目的长期目标是阐明调节突触接触形成的分子机制。突触是神经系统形成和功能的核心组成部分，在各种精神和神经系统疾病中受到影响。因此，发现参与突触连接建立的分子和机制将有助于我们理解神经元回路的形成，精神和神经疾病的原因，并可能确定治疗干预的潜在目标。突触的形成是一个程序良好的发育过程，涉及由不同分子群进行的各种细胞-细胞相互作用，这些相互作用是识别突触后靶标、突触膜之间接触的稳定以及突触间室之间功能偶联所必需的。目前的建议侧重于鉴定和表征的蛋白质能够诱导神经元之间的突触接触形成在胚胎发生。我们的中心假设是，在诱导突触前末端分化和突触接触建立的过程中，细胞膜系索或突触后神经元分泌配体的表达是必需的。因此，我们进行了全基因组搜索，寻找在突触发育的不同阶段突触后神经元中表达的基因转录本。该分析鉴定出一组含有免疫球蛋白（Ig）结构域、富亮氨酸重复序列（LRR）或其他蛋白质相互作用结构域的蛋白质，这些蛋白质在突触形成的起始阶段大幅上调，并且具有细胞表面配体的分子特征，表明这些蛋白质可能具有突触形成活性。在这个为期两年的项目中，我们提出：1)实验测试在突触形成起始过程中上调的这一部分蛋白的突触形成活性；2)研究它们的组合表达是否需要触发突触形成；3)测试GPI连接的细胞粘附分子的细胞表面表达是否调节烟碱乙酰胆碱神经递质受体的表面表达水平。为此，体外细胞试验将通过评估突触前终末分化和细胞表面蛋白表达水平来确定这些蛋白的突触生成活性。鉴定体外足以诱导突触形成的蛋白质将在未来进一步研究，以确定这些蛋白质是否为体内突触形成的起始所必需，并检查所涉及的分子机制。公共卫生相关性：该项目的长期目标是阐明参与突触接触组装的细胞和分子机制。目前的建议侧重于鉴定和表征基因和蛋白质参与突触形成的起始阶段。中心假设是，在假定的突触后神经元表面表达的蛋白质对于诱导接触部位的突触前末端的分化是必要的。了解突触形成的分子机制的重要性被以下事实所强调：突触是神经元交流的中心，并在包括智力发育障碍、自闭症和精神分裂症在内的各种精神和神经疾病中受到影响。因此，在本应用程序中提出的研究将导致更好地理解参与突触连接形成的分子机制，阐明精神和神经疾病的原因，并确定治疗策略。

DESCRIPTION (provided by applicant): The long-term goal of this research project is to elucidate the molecular mechanisms that regulate the formation of synaptic contacts. Synapses are a central component in the formation and the functioning of the nervous system and become affected in a variety of mental and neurological disorders. Therefore the discovery of the molecules and mechanisms that participate in the establishment of synaptic connections will contribute to our understanding of the formation of neuronal circuits, the causes of mental and neurological disorders, and may identify potential targets for therapeutic interventions. Synapse formation is a well-programmed developmental process that involves a variety of cell-cell interactions carried out by distinct groups of molecules, which are required for the recognition of a postsynaptic target, the stabilization of the contact between synaptic membranes, and the functional coupling between synaptic compartments. The present proposal focuses on the identification and characterization of proteins capable of inducing the formation of synaptic contacts between neurons during embryogenesis. Our central hypothesis is that the expression of membrane tethered or secreted ligands by the presumptive postsynaptic neuron are required to induce the differentiation of the presynaptic terminal and the establishment of a synaptic contact. For this reason, we carried out a genome-wide search for gene-transcripts that become expressed in postsynaptic neurons during the different phases of synapse development. This analysis led to the identification of a group of proteins containing immunoglobulin (Ig) domains, leucine-rich repeats (LRR), or other protein interacting domains that are substantially up-regulated during the initiation phase of synapse formation, and which have the molecular features of cell surface ligands suggesting that these proteins may possess synaptogenic activity. In this two-year project we propose 1) to test experimentally the synaptogenic activity of this subset of proteins up-regulated during the initiation of synapse formation, 2) to examine whether their combinatorial expression is needed to trigger synapse formation, and 3) to test whether cell surface expression of GPI- linked cell adhesion molecules regulates the surface expression levels of nicotinic acetylcholine neurotransmitter receptors. To this aim, in vitro cell assays will be used to determine the synaptogenic activity of these proteins by evaluating presynaptic terminal differentiation and cell surface protein expression levels. The identification of proteins which are sufficient to induce synapse formation in vitro will be further studied in the future to determine whether these proteins are necessary for the initiation of synapse formation in vivo and to examine the molecular mechanisms involved. PUBLIC HEALTH RELEVANCE: The long-term goal of this project is to elucidate the cellular and molecular mechanisms that participate in the assembly of synaptic contacts. The present proposal focuses on the identification and characterization of genes and proteins that participate in the initiation phase of synapse formation. The central hypothesis is that proteins expressed on the surface of presumptive postsynaptic neuron are necessary for inducing the differentiation of a presynaptic terminal at the site of contact. The importance of understanding the molecular mechanisms of synapse formation is underscored by the fact that synapses are the center piece for neuronal communication and become affected in a variety of mental and neurological disorders including, intellectual developmental disabilities, autism, and schizophrenia. Thus, the studies proposed in this application will lead to a better understanding of the molecular mechanisms that participate in the formation of synaptic connections, to the elucidation of the causes of mental and neurological disorders, and to the identification of therapeutic strategies.