Dynamic control of synaptic substructure and function by adhesion molecules

信息编码、存储和检索等复杂的神经过程是由精确和 突触强度的有效调节。对突触传递机制的研究将不会提供 不仅是我们如何看待神经回路的功能，还有我们如何更好地治疗神经生物学疾病， 在基础层面上的障碍。我们的实验室最近发现了一种突触下结构的新元素， 该受体激活可以独立于常规机制而被调节。蛋白质建立 神经递质胞吐作用的突触前位点在突触上与突触后位点紧密排列， 受体的纳米簇。这种跨突触结构的“纳米柱”预计将影响突触 通过控制受体活化的可能性来提高功效（Tang等，2016）。然而，尽管详细 检查受体如何在突触内和周围移动，我们几乎完全缺乏了解， 这些机制决定了它们在突触内和释放位点之间的位置。虽然 许多机制可能有助于纳米柱的形成，一个特别有吸引力的模型是突触细胞 粘附分子（CAM）通过高亲和力跨突触蛋白结合介导排列。我的目标是 来测试这个想法。然而，区分CAMs在突触中的持续作用， 突触形成是困难的。突触CAM经历广泛的剪接，并包括各种蛋白质 具有类似的功能域，在随后的几天里引发了广泛的机械补偿 击倒或击倒。为了避免这些影响，我一直在开发一种方法， 跨突触结合的时间尺度只有几分钟。我初步的资料本是采用办法的 由Peixoto等人（2012）通过将蛋白酶切割位点插入到感兴趣的蛋白质中， 所需蛋白质结构域的急性和特异性切割。我的设计包括一个可拆卸的替代品 策略，允许独立跟踪切割的组分，并且可以扩展到靶向多个 蛋白质同时在这里，我建议应用我的方法来测试突触CAM是否富含亮氨酸， 重复跨膜神经元2（LRRTM 2）介导突触纳米排列。LRRTM 2是一个强大的 因为它参与了与关键蛋白（突触后PSD-95）的跨突触结合 和突触前neurexin），它调节突触发生，其敲低导致诱发的 EPSC。有趣的是，与大多数其他CAM不同，LRRTM 2还直接结合突触后神经元内的AMPAR。 密度的通过膜片钳电生理学、超分辨率显微镜和单分子跟踪，我将 使用急性切割来测试LRRTM 2细胞外相互作用的消除是否急性破坏反式- 突触蛋白排列、AMPA受体迁移率和突触强度。这些结果将是第一次测试， 一个重要的新的突触机制，并将提供关键的培训，建立基础， 博士后工作

The complex neural processes of information encoding, storage, and retrieval are enabled by precise and efficient regulation of synaptic strength. Investigation into mechanisms of synaptic transmission will inform not only how we think about neural circuit functions, but also how we can better treat neurobiological diseases and disorders at a fundamental level. Our lab recently discovered a novel element of subsynaptic structure by which receptor activation may be modulated independent of conventional mechanisms. Proteins that establish presynaptic sites of neurotransmitter exocytosis are tightly aligned across the synapse with postsynaptic nanoclusters of receptors. This “nanocolumn” of trans-synaptic structure is expected to impact synaptic efficacy by controlling the likelihood of receptor activation (Tang et al., 2016). However, despite much detailed examination of how receptors move in and around synapses, we almost completely lack understanding of the mechanisms that determine their positioning within the synapse and across from sites of release. Though many mechanisms may contribute to nanocolumn formation, a particularly attractive model is that synaptic cell adhesion molecules (CAMs) mediate alignment through high affinity trans-synaptic protein binding. My goal is to test this idea. However, distinguishing the ongoing roles of CAMs at synapses following their known roles in synaptogenesis is difficult. Synaptic CAMs undergo extensive splicing and include a large variety of proteins with similar functional domains, provoking widespread mechanistic compensation over the days following knockout or knockdown. To avoid these effects, I have been developing approaches to acutely perturb CAM trans-synaptic binding on the time scale of just minutes. My preliminary data adapts an approach originally developed by Peixoto et al. (2012) by inserting a protease cleavage site into the protein of interest, enabling acute and specific cleavage of desired protein domains. My design includes a knockdown-replacement strategy, permits independent tracking of the cleaved components, and can be expanded to target multiple proteins simultaneously. Here, I propose to apply my approach to test whether the synaptic CAM Leucine-Rich Repeat Transmembrane neuronal 2 (LRRTM2) mediates synaptic nanoalignment. LRRTM2 is a strong candidate to test first because it participates in trans-synaptic binding with key proteins (postsynaptic PSD-95 and presynaptic neurexin), it regulates synaptogenesis, and its knockdown results in decreased evoked EPSCs. Intriguingly, unlike most other CAMs, LRRTM2 also directly binds AMPARs within the postsynaptic density. With patch-clamp electrophysiology, super-resolution microscopy, and single-molecule tracking, I will use acute cleavage to test whether elimination of LRRTM2 extracellular interactions acutely disrupts trans- synaptic protein alignment, AMPA receptor mobility, and synaptic strength. These results will be the first test of an important new synaptic mechanism, and will provide key training establishing the basis for subsequent postdoctoral work.