Neuroligin Regulation of Central GABAergic Synapses

描述（由申请人提供）：本R03提案的目标是确定细胞粘附分子（CAM）神经素2 （NL2）调节抑制性突触传递的分子机制。虽然已经确定神经胶质素介导突触生成活动，但尚不清楚神经胶质素是否也参与突触形成后功能性调节突触传递的事件。在我们的初步研究中，在培养的神经元和表达NL2和GABAA受体（GABAARs）的HEK293细胞共培养系统中，NL2足以诱导gaba能突触的形成。此外，NL2过表达诱导培养的小脑颗粒细胞（cgc）中微型抑制性突触后电流（mIPSCs）的频率和峰值幅度显著增加。引人注目的是，在NL2过表达的CGCs中，mIPSCs的衰减速度明显快于对照CGCs。我们和其他人已经证明，GABAA Rs的1个亚基从12/3切换到11是中枢神经系统各区域IPSC衰变加速的基础。这些结果导致了我们的主要假设，即NL2通过在突触后位点招募11个含有GABAA受体的亚基来促进GABAA能突触的成熟并加速突触的衰退，并且这种招募是通过NL2相互作用的支架蛋白S- SCAM进行的。为了验证这一假设，我们将追求三个具体目标：(1)确定GABAAR亚基组成的改变是否导致了NL2诱导的GABAAR能突触电流衰减时间的加速；(2)确定NL2的胞内结构域，该结构域负责在突触后位点招募11个含有GABAARs的亚基；(3)确定NL2主要相互作用蛋白S-SCAM在NL2在突触后位点募集11个GABAARs中的作用。突触的发育是大脑神经元回路形成的关键过程。异常的突触发育和突触功能障碍可能导致严重的神经发育障碍，如自闭症、阿斯伯格综合征和智力低下。这些研究将使我们了解神经脂素如何调节抑制性突触传递，因此，有助于我们进一步了解神经脂素在突触中的功能作用。由于神经发育障碍的病因复杂，临床症状多样，其治疗仍远不令人满意。阐明神经胶质素在抑制性突触中的功能作用无疑将增加我们治疗这类疾病的能力。公共卫生相关性：突触的形成和成熟是大脑神经元回路生成的关键过程。异常的突触发育和突触功能障碍可能导致神经发育障碍，如自闭症、阿斯伯格综合征和智力低下。这些疾病的特征是社会互动受损、沟通缺陷和重复行为。由于发育障碍的病因复杂，临床症状多样，目前的诊断和治疗技术还远远不能令人满意。随着神经科学研究的迅速发展，人们清楚地认识到，所有的神经发育障碍都在一个可能的部位发生相应的功能改变：突触及其发育。最近的研究结果揭示了神经素（NLs）成员在突触形成中的作用。NLs是表达于突触后膜表面的突触后细胞粘附分子（CAMs）。nl在突触前神经末梢与它们的突触前伙伴神经素结合。nl /神经素之间的配对跨越突触间隙，形成物理链。nl在突触发生中的作用可能不像先前预期的那样局限于兴奋性突触。NLs的下调导致兴奋性和抑制性突触的缺失。事实上，NL2， NLs家族成员之一，被发现在抑制性突触富集。我们和其他人已经证明，NL2促进gaba能突触形成的初始步骤，可能是通过与与转染的HEK293细胞接触的轴突突上表达的神经素的跨突触相互作用。然而，一个重要的问题仍然存在：nl是否在突触形成后调节突触传递？我们惊人的初步研究表明，在NL2过表达培养的小脑颗粒细胞（CGCs）中，mIPSCs的衰变明显快于对照CGCs。这些结果支持了我们的主要假设，即NL2通过招募具有快速衰减时间成分的突触GABAA受体来促进GABAA能成熟并加速突触传递。为了验证这一假设，本研究将追求三个目标：(1)确定GABAAR亚基组成的改变是否导致了NL2诱导的GABAAR能突触电流衰减时间的加速；(2)确定NL2的胞内结构域，该结构域负责在突触后位点招募11个含有GABAARs的亚基；(3)确定NL2主要相互作用蛋白S-SCAM在NL2在突触后位点募集11个GABAARs中的作用。我们相信，我们提出的上述研究将使我们了解神经脂素如何调节抑制性突触传递，因此，有助于我们进一步了解神经脂素在突触中的功能作用。虽然神经发育障碍明显有很强的遗传成分，但其复杂的病因和各种临床症状使神经发育障碍的治疗远远不能令人满意。阐明神经节素在抑制性突触中的功能作用，无疑将增加我们开发更有效的诊断和治疗方法的能力。

DESCRIPTION (provided by applicant): The goal of this R03 proposal is to identify molecular mechanisms underlying the regulation of inhibitory synaptic transmission by the cell adhesion molecule (CAM) neuroligin 2 (NL2). Although it has been established that neuroligins mediate synaptogenic activity, it is not known if neuroligins also participate in events that functionally regulate synaptic transmission once the synapse is formed. In our preliminary studies, NL2 was sufficient to induce GABAergic synapse formation in a co-culture system between cultured neurons and HEK293 cells expressing NL2 and GABAA receptors (GABAARs). In addition, NL2 over-expression induces a significant increase in frequency and peak amplitude of miniature inhibitory postsynaptic currents (mIPSCs) in cultured cerebellar granule cells (CGCs). Strikingly, the decay of mIPSCs in the NL2 overexpressing CGCs is significantly faster than that seen in control CGCs. We and others have shown that the switch of 1 subunits of GABAA Rs from 12/3 to 11 underlies developmental speeding of the IPSC decay in various CNS regions. These results lead to our main hypothesis that NL2 facilitates GABAergic synapse maturation and accelerates synaptic decay by recruiting 11 subunit containing GABAA receptors at postsynaptic sites, and that this recruitment occurs via the NL2 interacting scaffolding protein S- SCAM. To test this hypothesis, we will pursue three specific aims: (1) To determine whether altered GABAAR subunit composition accounts for the acceleration in decay time of GABAergic synaptic currents induced by NL2; (2) To define the intracellular domains of NL2 that are responsible for recruiting 11 subunit containing GABAARs at postsynaptic sites; (3) To determine the role of the S-SCAM, the main NL2 interacting protein, in recruitment of 11 containing GABAARs at postsynaptic sites by NL2. Synapse development is a crucial process in the generation of neuronal circuits in the brain. Aberrant synapse development and synaptic dysfunction may lead to severe neurodevelopmental disorders, such as autism, Asperger syndrome and mental retardation. These studies will enable us to understand how neuroligins regulate inhibitory synaptic transmission, and, therefore, serve to further our understanding of the functional role of neuroligins at synapses. Because of their complicated etiologies and various clinical symptoms, treatments of neurodevelopmental disorders are still far from satisfaction. Elucidating functional role of neuroligins at inhibitory synapses will no doubt increase our ability to treat this group of diseases. PUBLIC HEALTH RELEVANCE: Synapse formation and maturation are crucial processes in the generation of neuronal circuits in the brain. Aberrant synapse development and synaptic dysfunction may lead to neurodevelopmental disorders such as autism, Asperger syndrome and mental retardation. These diseases are characterized by impaired social interaction, communication deficits, and repetitive behaviors. Because of their complicated etiologies and various clinical symptoms, techniques of diagnosis and treatment of development disabilities are still far from satisfaction. With the rapid development in neuroscience research, it becomes clear that all neurodevelopmental disorders have corresponding functional changes in one possible site: the synapse and its development. Recent findings have shed some new light on the involvement of members of the neuroligins (NLs) in synapse formation. NLs are postsynaptic cell adhesion molecules (CAMs) expressed on the surface of postsynaptic membrane. NLs bind to their presynaptic partners, neurexins, on presynaptic nerve terminals. The pairing between NLs/neurexins spans the synaptic cleft and forms a physical tether. The function of NLs in synaptogenesis might not be restricted to excitatory synapses as previously anticipated. Down-regulation of NLs results in a loss of both excitatory and inhibitory synapses. Indeed, NL2, one of the family members of NLs, is found enriched at inhibitory synapses. We and others have shown that NL2 promotes the initial steps in GABAergic synapse formation, presumably via trans-synaptic interactions with neurexins expressed on axonal processes in contact with transfected HEK293 cells. However, one important question is still remained: do NLs regulate synaptic transmission once the synapse is formed? Our striking preliminary studies demonstrated that the decay of mIPSCs in the NL2 overexpressing cultured cerebellar granule cells (CGCs) is significantly faster than that seen in control CGCs. These results lead to our main hypothesis that NL2 facilitates GABAergic maturation and accelerates synaptic transmission by recruiting synaptic GABAA receptors with fast decay time component. To test this hypothesis, threes aims will be pursued in this current proposal: (1) To determine whether altered GABAAR subunit composition accounts for the acceleration in decay time of GABAergic synaptic currents induced by NL2; (2) To define the intracellular domains of NL2 that are responsible for recruiting 11 subunit containing GABAARs at postsynaptic sites; (3) To determine the role of the S-SCAM, the main NL2 interacting protein, in recruitment of 11 containing GABAARs at postsynaptic sites by NL2. We believe that the studies we proposed above will enable us to understand how neuroligins regulate inhibitory synaptic transmission, and, therefore, serve to further our understanding of the functional role of neuroligins at synapses. Though it is clear that there is a strong genetic component to neurodevelopmental disorders, their complicated etiologies and various clinical symptoms make the treatments of neurodevelopmental disorders far from satisfaction. Elucidating functional role of neuroligins at inhibitory synapses will no doubt increase our ability to develop more effective diagnosis and treatment methods for this group of diseases.