The molecular architecture of perineuronal nets

神经元周网（PNN）是一种引人注目的神经细胞外基质（ECM）结构， 在过去的十年中，他们在神经发育可塑性中发挥的关键作用引起了人们的极大兴趣。这些 复杂的大分子结构与一系列认知功能有关，并在各种不同的情况下发生变化。 神经系统紊乱的症状尽管人们对PNN功能的兴趣越来越大，但它们的作用机制 调节神经功能知之甚少，因为目前还没有工具或技术来 具体操作PNN。我们推测，我们无法瞄准和破坏PNN主要是由一个 缺乏对其分子组成或结构的了解。我们在这个提案中的目标是进行 已知PNN组分的结构-功能分析以及鉴定锚网至神经元的蛋白质 表面。使用体外和体内方法的强大组合，我们已经获得了强有力的初步结果。 数据详细说明了新发现的PNN组分受体蛋白酪氨酸磷酸酶ζ（RPTPζ） 在分子水平上与PNN内的生腱蛋白-R（TNR）缔合。此外，我们的数据表明， RPTP β·TNR复合物通过GPI连接的蛋白质接触蛋白1（CNTN 1）将PNN锚定到神经元细胞表面， 这使得CNTN 1成为第一个被鉴定的PNN表面结合蛋白。我们的核心假设是， PNN有一组独特的成分和受体，它们使PNN成核并将它们锚到特定的 神经元细胞表面，从而定义其独特的结构和功能。本提案的总体目标是 识别PNN特异性成分，并通过以下独特的组合来剖析PNN的形成： 邻近标记分析，蛋白质结合分析和蛋白质X射线晶体学，以创建工具 来针对和精确地操纵这些结构。我们的长期目标是利用这些工具 剖析PNN的功能，以便更好地了解疾病的发病机制，并最终靶向PNN 治疗上在我们强有力的初步数据的指导下，这项提案旨在发现独特的成分， 通过追求三个不重叠的具体目标来指导PNN的组装：1）定义 RPTP β·TNR复合物在PNNs锚定神经元表面中的作用; 2）研究PNNs的生物化学和结构特征， 表征ACAN、HAPLN 1和TNR之间的相互作用;以及3）鉴定细胞表面受体， PNN的新组件。拟议的工作是重要的，因为它将试图确定关键的独特的 这些成分有助于PNN的形成并因此发挥功能。圆满完成目标 将提供关键的见解和试剂，以具体和精确地操纵PNN，并最终了解 其功能机制。这种方法是创新的，因为它汇集了一种新颖的组合， 生理学、生物化学和结构学方法来研究这些重要的大分子组装体 在中枢神经系统中。最终，拟议的工作可能会对该领域产生变革性影响， PNN在健康和疾病中的功能。

Perineuronal nets (PNNs) are conspicuous neural extracellular matrix (ECM) structures that have garnered significant interest over the last decade for the critical roles they play in neural developmental plasticity. These complex macromolecular structures are implicated in an array of cognitive functions, and are altered in a variety of neurological disorders. Despite the growing interest in PNN functions, the mechanisms by which they modulate neural functions are poorly understood, because there are currently no tools or techniques to manipulate PNNs specifically. We surmise that our inability to target and disrupt PNNs is primarily driven by a lack of understanding of their molecular composition or structure. Our goal in this proposal is to conduct a structure-function analysis of known PNN components as well as to identify proteins that anchor nets to neuronal surfaces. Using a powerful combination of in vitro and in vivo approaches, we have obtained strong preliminary data detailing how the newly identified PNN component receptor protein tyrosine phosphatase zeta (RPTPζ) associates with tenascin-R (TNR) within PNNs at a molecular level. Furthermore, our data indicate that the RPTPζ•TNR complex anchors PNNs to the neuronal cell surface via the GPI-linked protein contactin-1 (CNTN1), which makes CNTN1 the first surface binding protein for PNNs ever identified. Our central hypothesis is that there are a set of unique components and receptors of PNNs that nucleate PNNs and anchor them to specific neuronal cell surfaces, thereby defining their unique structure and functions. The overall objective of this proposal is to identify PNN-specific components and dissect the formation of PNNs through a unique combination of proximity-labeling assays, protein-binding assays, and protein X-ray crystallography in order to create the tools to target and manipulate these structures specifically and precisely. Our long-term goal is then to use these tools to dissect PNN function in order to better understand disease pathogenesis and ultimately to target PNNs therapeutically. Guided by our strong preliminary data, this proposal seeks to discover the unique components that guide the assembly of PNNs by pursuing three non-overlapping specific aims: 1) defining the role of the RPTPζ•TNR complex in anchoring PNNs to neuronal surfaces; 2) pursuing the biochemical and structural characterization of interactions between ACAN, HAPLN1, and TNR; and 3) identifying cell surface receptors and novel components of PNNs. The proposed work is significant because it will attempt to identify the key unique components that contribute to the formation and thereby function of PNNs. Successful completion of the aims will provide key insights and reagents to manipulate PNNs specifically and precisely and ultimately understand their functional mechanisms. This approach is innovative because it brings together a novel combination of physiological, biochemical and structural approaches to investigate these important macromolecular assemblies in the central nervous system. Ultimately, the proposed work could be transformative for the field and lead to key mechanistic insights into of PNN function in health and disease.