Trans-synaptic mechanism of retinal synapse formation and function

项目摘要/摘要 我们最佳的视觉表现始于视网膜中正确的并行视觉信息处理 依赖于精确的视网膜神经回路的形成。要做到这一点，视网膜神经元必须准确地设置 功能性突触需要在突触前神经递质释放部位之间精确对准 以及突触后受体。越来越多的证据表明，细胞黏附分子(CAM)介导的 跨突触复合体对于突触的形成和功能是至关重要的，然而，如何前-突触复合体的机制？ 突触释放机制与视网膜带突触后受体信号复合体协调 突触在很大程度上是未知的。我们最近报道了细胞外钙通道辅助亚单位 α-2-δ-4是视杆感受器轴突形成和突触形成所必需的，并证明了 α2δ4通过控制富含亮氨酸的重复蛋白ELFN1的突触靶向来实现这一点 在杆状突触特异表达，并与突触后mGluR6受体跨突触相互作用。 我们还证明了α2δ4通过LRR域与ELFN1相互作用，LRR域是一个共享的保守结构域 许多LRR蛋白。有趣的是，我们和其他实验室发现了另一种名为LRIT1的LRR蛋白，它 特异地影响锥体突触功能。拟议工作的目标是确定它是否是一个 突触前钙通道复合体利用α-2-δ-4与突触后协同的一般机制 受体通过不同的LRR蛋白的促进作用。在目标1中，我们将确定 α-2、δ-4-ELFN1在体内电穿孔结合缺失的视杆突触形成中的相互作用 诱变。在目标2中，我们将通过测试LRIT1是否 使用电生理记录通过α2δ4影响突触前Cav1.4通道的活动。在《目标3》中，我会 研究内丛状层(IPL)的突触是否也采用类似的跨突触机制 用该技术研究α_2、δ_4和LRIT_1对红细胞无长突触形成和功能的影响 在指导阶段获得的。这一建议是创新的，因为没有进行任何机械性的研究 在α-2，δ-4蛋白上，尽管它在光感受器突触的形成和功能中起着重要的作用。这个 拟议的工作意义重大，因为这项研究的结果将使我们能够更好地了解视网膜是如何 神经元建立和维持突触联系。

PROJECT SUMMARY/ABSTRACT Our optimal visual performance starts with the correct parallel visual information processing in the retina which depends on precise retinal neural circuit formation. To achieve this, retinal neurons must accurately set up functional synapses which require precise alignment between the pre-synaptic neurotransmitter releasing site and the post-synaptic receptor. Increasing evidence suggests that cell adhesion molecules (CAMs)-mediated trans-synaptic complexes are critical for synapse formation and function, however, the mechanism how pre- synaptic releasing machinery coordinates with the post-synaptic receptor signaling complex at retinal ribbon synapses is largely unknown. We recently reported that the extracellular calcium channel auxiliary subunit α2δ4 is necessary for rod photoreceptor axonal elaboration and synapse formation and demonstrated that α2δ4 does so through controlling the synaptic targeting of ELFN1, a leucine-rich repeat (LRR) protein specifically expressed at rod synapse and interacts trans-synaptically with the post-synaptic mGluR6 receptor. We also showed that α2δ4 interacts with ELFN1 through the LRR domain, a conserved domain shared across many LRR proteins. Interestingly, we and other lab identified another LRR protein termed LRIT1, which specifically affects cone synaptic function. The objective of the proposed work is to determine whether it is a general mechanism that pre-synaptic calcium channel complex utilizes α2δ4 to coordinate with post-synaptic receptor through the facilitation of different LRR proteins. In Aim 1, we will determine the functional role of α2δ4-ELFN1 interaction in rod synapse formation using in vivo electroporation combined with deletion mutagenesis. In Aim 2, we will study how LRIT1 regulates cone synaptic function by testing whether LRIT1 affects pre-synaptic Cav1.4 channel activity through α2δ4 using electrophysiological recording. In Aim 3, I will investigate whether synapses in the inner plexiform layer (IPL) also adopt similar trans-synaptic mechanism by studying how α2δ4 and LRIT1 affect RBC-AII amacrine synapse formation and function using the technique gained during the mentored phase. This proposal is innovative because no mechanistic studies has been done on α2δ4 protein despite its clearly important role in photoreceptor synapse formation and function. The proposed work is significant since results from this study will enable better understanding of how retinal neurons establish and maintain synaptic contact.