Proj 2 - Lymphatic system as a conduit for age-related defective proteostasis in AD

在小鼠中观察到伴随细胞蛋白质稳态变化的促炎特征， 脑神经退行性疾病，如AD，以及在较低程度上在老化的脑中。但尽管 对炎症，AD发展和炎症之间联系的理解有了很大的进步。 关于衰老相关的病理学，有几个重要的方面仍在研究之中。 例如，尽管认识到淋巴系统的存在，但作为大脑到外周的 尽管淋巴管是一个重要的沟通管道，但淋巴在炎症/退行性疾病中所起的作用仍然是未知的。 发生在脑实质中并将脑分子特征输送到颈部的过程 免疫监视的节点另一个重要的方面，尚待调查，是非常早期的变化， 内体蛋白质稳态和蛋白质组翻译后修饰，发生在真正的AD之前， 观察到老化退行性变化。最后，尽管主动和被动免疫疗法 已经提出，对于AD，MHCII限制性免疫应答对自然加工的TAU、Aβ和其他免疫应答的作用是可能的。 大脑自身抗原仍然未知。因此，本申请的目的是：（一）探讨 淋巴循环运输脑的炎症/变性分子标记 到引流颈淋巴结的实质（具有P1和核心B）（ii）映射大脑中的非常早期的变化 蛋白质组、PTM修饰、内溶酶体蛋白质稳定和自噬机制（含P1和核心C） (iii)分析衰老过程中MHC II限制性免疫肽组和相关T细胞反应的变化 以及AD进展的不同阶段（P3和核心B）。通过使用最先进的定量 蛋白质组学与细胞生物学方法相关，我们将绘制衰老不同阶段的蛋白质PTM 过程和AD的发展以及它们对内溶酶体蛋白质稳态和自噬的影响 机械.此外，我们还将研究年轻人和老年人的淋巴炎性/退行性特征， 小鼠以及处于不同疾病阶段的AD小鼠。最后，将MHC II-免疫肽组，从 将通过MS/MS分析颈淋巴结中的树突状细胞，以绘制来自脑相关的 蛋白质在衰老和AD的不同阶段。将使用相关MHC-II肽进行四聚体染色， 用于分析T细胞识别并解决对脑抗原的整体免疫反应。 这个项目的所有结果将提供一个渐进的快照，细胞蛋白质组是如何 在AD或衰老发展的早期到晚期阶段，自噬机制如何参与 在处理修饰的蛋白质组时，这些早期变化如何逐渐发展成复杂的聚集体 以及内溶酶体系统如何参与恢复蛋白质稳态。此外，淋巴细胞如何 系统作为一个管道，运输与AD相关的炎症/退行性表型 免疫系统的发展，以及相反，免疫细胞如何应对衰老中的蛋白质组变化 在AD进展期间。

Summary A pro-inflammatory signature accompanied by changes in cellular proteostasis are observed in brain neurodegenerative conditions such as AD and to less extent in the aging brain. However, despite the great advancement towards the understanding of the connection between inflammation, AD development and aging-related pathologies, there are several important aspects, which are still very much under investigated. For example albeit it is appreciate the existence of the lymphatic system, as a brain-to-periphery communication conduit, it is still unknown the role played by the lymph in the inflammatory/degenerative process occurring in the brain parenchyma and in transporting the brain molecular signature to the cervical nodes for immune-surveillance. Another important aspect, yet to be investigated, are the very early changes in endosomal proteostasis and the proteome post-translational modifications, occurring before bona fide AD and aging degenerative changes are observed. Finally, despite the fact that active and passive immunotherapy has been proposed for AD the role of MHCII-restricted immune response to naturally processed TAU, Aβ and other brain self-antigens is still unknown. As such the trust of this application is to: (i) explore the role played by the lymphatic circulation in transporting the inflammatory/degenerative molecular signature of the brain parenchyma to the draining cervical node (with P1 and Core B) (ii) map the very early changes in the brain proteome, PTM- modifications, endo-lysosomal proteostasis and autophagic machinery (with P1 and Core C) (iii) analyze changes in the MHC II-restricted immune-peptidome, and related T cell responses, during aging and the different phases of AD progression (with P3 and Core B). By using state-of the art quantitative proteomic, associated with a cell biology approach, we will map proteins PTMs at different stages of the aging process and AD development as well as their effect on endo-lysosomal proteostasis and the autophagic machinery. Additionally we will investigate the lymph inflammatory/degenerative signature in young and old mice, as well as, AD mice at different stage of disease. Finally, the MHC II-immunopeptidome, eluted from dendritic cells in the cervical node, will be analyzed by MS/MS to map peptides derived from brain-relevant proteins in aging and at different stages of AD. Tetramer staining, using relevant MHC-II-peptides, will be employed to analyze T cell recognition and address the overall immune responses to brain antigens. Altogether results from this project will provide a progressive snap shot of how the cellular proteome is modified during the early-to-late stages of AD or aging development, how the autophagic machinery is involved in disposing the modified proteome, how these early changes progressively develop into complex aggregates and how the endo-lysosomal system is involved in restoring proteostasis. Additionally, how the lymphatic system function as a conduit to transport the inflammatory/degenerative phenotype associated with AD development to the immune system and conversely how immune cells respond to proteomic changes in aging and during AD progression.