Neuroligin Regulation of Central GABAergic Synapses

描述（由申请人提供）：本R 03提案的目标是确定细胞粘附分子（CAM）神经配素2（NL 2）调节抑制性突触传递的分子机制。虽然已经确定神经配蛋白介导突触发生活性，但尚不清楚神经配蛋白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NL 2和GABAA受体（GABAAR）的HEK 293细胞之间的共培养系统中，NL 2足以诱导GABA能突触形成。此外，NL 2过表达诱导培养的小脑颗粒细胞（CGC）中的微型抑制性突触后电流（mIPSC）的频率和峰值幅度显著增加。引人注目的是，在NL 2过表达的CGC中mIPSC的衰变比在对照CGC中观察到的显著更快。我们和其他人已经表明，GABAA Rs的1亚基从12/3到11的转换是各种CNS区域中IPSC衰变的发育加速的基础。这些结果导致我们的主要假设，即NL 2通过在突触后位点募集含有GABAA受体的11亚基来促进GABA能突触成熟并加速突触衰退，并且这种募集通过NL 2相互作用的支架蛋白S-SCAM发生。为了验证这一假设，我们将进行三个具体的目标：（1）确定改变GABAAR亚单位组成是否解释了由NL 2诱导的GABA能突触电流衰减时间的加速：（2）确定负责在突触后位点募集含11个GABAAR亚单位的NL 2细胞内结构域;（3）研究NL 2的主要相互作用蛋白S-SCAM在NL 2募集11个含GABAAR的突触后位点中的作用。突触发育是大脑神经元回路产生的关键过程。异常的突触发育和突触功能障碍可导致严重的神经发育障碍，如自闭症、神经发育障碍综合征和智力低下。这些研究将使我们能够了解神经连接素如何调节抑制性突触传递，因此，有助于进一步了解神经连接素在突触中的功能作用。神经发育障碍病因复杂，临床症状多样，治疗效果不理想。阐明神经配素在抑制性突触中的功能作用无疑将提高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产生的关键过程。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、神经发育障碍综合征和智力低下。这些疾病的特征是社会互动受损，沟通障碍和重复行为。发育性残疾病因复杂，临床症状多样，其诊断和治疗技术还很不完善。随着神经科学研究的迅速发展，越来越清楚的是，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变化：突触及其发育。最近的研究结果揭示了一些新的光参与的神经胶质（NLs）的成员在突触的形成。NLs是表达于突触后膜表面的突触后细胞粘附分子（CAMs）。NLs在突触前神经末梢上与它们的突触前伙伴神经毒素结合。NL/neurexins之间的配对跨越突触间隙并形成物理系链。NLs在突触发生中的功能可能并不像以前预期的那样仅限于兴奋性突触。NL的下调导致兴奋性和抑制性突触的损失。事实上，发现NL 2（NL的家族成员之一）在抑制性突触处富集。我们和其他人已经表明，NL 2促进GABA能突触形成的初始步骤，推测是通过与轴突突起上表达的neurexins的跨突触相互作用，轴突突起与转染的HEK 293细胞接触。然而，一个重要的问题仍然存在：一旦突触形成，NLs是否调节突触传递？我们惊人的初步研究表明，在NL 2过表达培养的小脑颗粒细胞（CGC）中，mIPSC的衰变明显快于对照CGC。这些结果导致我们的主要假设，即NL 2促进GABA能成熟，并通过招募具有快速衰减时间成分的突触GABAA受体来加速突触传递。为了验证这一假说，本研究拟从以下三个方面着手：（1）确定NL 2引起的GABA能突触电流衰减时间加快是否与GABAAR亚基组成的改变有关：（2）确定NL 2的胞内结构域，它负责在突触后部位募集含GABAAR的11个亚基;（3）研究NL 2的主要相互作用蛋白S-SCAM在NL 2募集11个含GABAAR的突触后位点中的作用。我们相信，我们提出的上述研究将使我们能够了解神经连接素如何调节抑制性突触传递，因此，有助于进一步了解神经连接素在突触中的功能作用。虽然神经发育障碍有很强的遗传成分，但其复杂的病因和多样的临床症状使得神经发育障碍的治疗远不能令人满意。阐明神经配素在抑制性突触中的功能作用无疑将提高我们为这组疾病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.