Control of Tripartite Synapse Formation by Astrocytic Neuroligins

 描述(申请人提供)：突触是中枢神经系统(CNS)的特殊细胞-细胞连接，对学习、记忆和认知至关重要。定位于这些连接的是连接突触前轴突和突触后树突的细胞黏附分子(CAM)，后者建立突触结构和功能。中枢神经系统突触现在被认为是一个由三个成员组成的实体，或称三部分突触，包括轴突终末、树突和包膜的星形胶质细胞突起。星形胶质细胞是非神经性的、结构复杂的细胞，其分支终止于数千个与突触相互作用的精细突起。一只小鼠星形胶质细胞一次可以接触超过10万个突触。虽然几十年的研究已经确定了两个神经元之间的突触是如何形成的，但调节星形胶质细胞-突触相互作用的分子机制仍然不清楚。此外，星形胶质细胞形态和突触联系之间的机制和分子联系尚不清楚。星形胶质细胞的分支复杂性在出生后第二周在啮齿动物的皮质中形成，这是一个重要的兴奋性突触形成的时期。在初步实验中，我发现星形胶质细胞在体外需要与神经元接触才能建立复杂的形态。因为星形胶质细胞专门接触突触，所以很可能是星形胶质细胞表达与这些神经元粘连相互作用的CaM。在初步实验中，我发现星形胶质细胞表达神经连接蛋白(NL)家族的CaM，众所周知，它在神经元突触中发挥关键作用，以前被认为只在神经元中起作用。NLS是被研究得很好的分子，它调节突触的形成和树突的结构。用shRNA敲除培养中的星形胶质细胞NLS表明，NLS是星形胶质细胞在神经元接触时建立形态所必需的。这些数据是新的，表明在星形胶质细胞中存在以前未知的NLS功能，这些功能可能将星形胶质细胞与突触联系起来，并控制复杂的星形胶质细胞结构。基于这些观察和先前发表的结果，这项提议将利用新的和成熟的技术来确定星形胶质细胞中的NLS是如何调节突触相互作用和产生星形胶质细胞结构复杂性的。通过结合细胞生物学、光学和电子显微镜以及电生理学，这项提案的目的是测试细胞的结构和功能要求 NLS在星形胶质细胞中的表达，并通过星形胶质细胞NLS确定星形胶质细胞-神经元接触和下游信号传递的分子机制。这一提议的结果将为研究神经元和星形胶质细胞在整个发育和神经可塑性过程中的相互作用开辟令人兴奋的新途径。NLS与许多疾病状态有关，包括自闭症和癫痫。因此，拟议的工作将阐明在三方突触的整体内重新评估这些凸轮的必要性。

 DESCRIPTION (provided by applicant): Synapses are specialized cell-cell junctions of the central nervous system (CNS) that are critical for learning, memory, and cognition. Localized to these junctions are cell adhesion molecules (CAMs) that link the presynaptic axon with the postsynaptic dendrite, which establish synaptic structure and function. CNS synapses are now considered a three-membered entity, or tripartite synapse, which include the axon terminal, the dendrite, and an ensheathing astrocyte process. Astrocytes are non-neuronal, architecturally complex cells with branches that terminate in thousands of fine processes that interact with synapses. A single mouse astrocyte can contact more than 100,000 synapses at a time. While decades of research have identified how synapses between two neurons are formed, the molecular mechanisms that regulate astrocyte-synapse interactions remain elusive. Additionally, the mechanisms and molecular links between astrocyte morphology and synapse association are unknown. Astrocyte branching complexity develops in the rodent cortex during the second postnatal week, a time of significant excitatory synapse formation. In preliminary experiments I found that astrocytes in vitro require contact with neurons to establish a complex morphology. Because astrocytes specifically contact synapses, it is likely that astrocytes express CAMs to interact with these neuronal adhesions. In preliminary experiments, I discovered that astrocytes express the neuroligin (NL) family of CAMs, which are known to play critical roles at neuronal synapses, and previously thought to be functional only in neurons. NLs are well-studied molecules that regulate synapse formation and structure of dendritic trees. Knockdown of astrocytic NLs in culture by shRNA demonstrated that NLs are required for establishing astrocyte morphology in response to neuronal contact. These data are novel and indicate that there are previously unknown functions of NLs in astrocytes, which potentially link astrocytes to synapses and control complex astrocyte architecture. Based on these observations and previously published results this proposal will utilize novel and well- established techniques to determine how NLs in astrocytes regulate synapse interaction and generate astrocyte architectural complexity. By combining cell biology, light and electron microscopy, and electrophysiology, the aims of this proposal will test the structural and functional requirement of NLs in astrocytes and identify the molecular mechanisms of astrocyte-neuron contact and downstream signaling through astrocytic NLs. The outcomes of this proposal will open exciting new avenues in studying the interactions between neurons and astrocytes throughout development and neural plasticity. NLs are associated with a number or disease states including autism and epilepsy. Therefore the proposed work will illuminate the need for reevaluation of these CAMs within the entirety of the tripartite synapse.