The Physical Biology of Neurodegeneration in Sporadic Amyotrophic Lateral Sclerosis/Frontotemporal Dementia

在散发性肌萎缩侧索硬化-额颞叶痴呆（sALS/FTD）临床谱中， 疾病相关蛋白如TDP-43的聚集和积累是一种值得注意的神经病理学变化。 然而，我们对为什么会发生这种高度异常的事件知之甚少。虽然在多个中断 细胞过程与ALS有关，但仍存在3个关键的知识空白：1）是什么触发了ALS， 在散发性疾病中野生型蛋白质的聚集？蛋白质聚集是否足以驱动病理学？（二） 是什么驱使细胞特异性的脆弱性和从ALS到FTD的可变临床表现？3)怎么 疾病相关的蛋白质稳态改变扰乱了组织微环境的通讯？ 鉴于超过95%的ALS是偶发性的，并且偶发性疾病的机制仍然存在， 未知，我们将超越个别突变，并建立一个新的概念框架，检查 在疾病状态下发生的细胞变化。我们通过关注TDP-43聚集的原因来解释这一点 发生，特别是在散发性ALS，我们将获得深入了解致病机制的基础上， 一系列的疾病我们的中心假设是细胞和组织发生了物理变化 引发ALS/FTD的规模。我们认为，改变细胞内的生物物理特性（主要是 改变的分子拥挤），这与组织微环境的机械扰动有关 （硬化，炎症，水肿引起渗透压），导致年龄依赖性细胞功能障碍， 改变大分子蛋白质机器的组装、拆卸和运输的动力学。我们将 在细胞模型、动物模型和患者组织中测试这一假设，方法是：（1）使用新的工具来探测 细胞的细胞内生物物理环境;（2）使用新的基因组学技术整合这些发现 应用于小鼠模型，研究（i）细胞内拥挤的变化和细胞外的变化， 组织微环境可能驱动体内发病机制，以及（ii）这种扰动如何破坏细胞-细胞 组织脆弱区域的通讯;（3）通过重新检查我们的发现与人类疾病相关 这些发现是在临床和神经病理学上深入策划的ALS/FTD患者队列的背景下进行的。 这些研究将使我们能够解决以下问题：1）为什么异常蛋白质聚集 和积累发生在散发性疾病，以及这可能如何有助于疾病的发病机制; 2） 这些蛋白质稳态的改变扰乱了组织微环境中的细胞间通讯; 和3）是什么驱动细胞类型的脆弱性，使ALS/FTD独特？拟议的工作将完成 以下内容：A）代表神经细胞中分子拥挤的第一次详细调查; B）揭示是否存在一种 细胞内拥挤、蛋白质聚集和神经变性之间存在因果联系，C）建立 细胞内拥挤的影响是否显示细胞类型特异性特征，包括蛋白质的变化， 蛋白质相互作用，和D）提供了一个新的框架，探索治疗ALS/FTD的治疗策略。

On the sporadic Amyotrophic Lateral Sclerosis-Frontotemporal Dementia (sALS/FTD) clinical spectrum, the aggregation and accumulation of disease-associated proteins such as TDP-43 is a notable neuropathological hallmark, yet we know little about why this highly abnormal event might occur. Although disruptions in multiple cellular processes have been implicated in ALS, 3 critical gaps in knowledge remain: 1) What triggers the aggregation of wildtype proteins in sporadic disease? Is protein aggregation sufficient to drive pathology? 2) What drives the cell-specific vulnerabilities and variable clinical manifestation from ALS to FTD? 3) How do disease-associated alterations in protein homeostasis perturb communication in the tissue microenvironment? Given that more than 95% of ALS arises sporadically, and that the mechanisms of sporadic disease remain unknown, we will look beyond individual mutations, and establish a novel conceptual framework that examines the cellular changes that occur during disease states. We posit that by focusing on why TDP-43 aggregation occurs, especially in sporadic ALS, we will gain insights into pathogenic mechanisms underlying this spectrum of disorders. Our central hypothesis is that there are physical changes at the cell and tissue scale that initiate ALS/FTD. We propose that altered biophysical properties within cells (predominantly altered molecular crowding), which are linked to mechanical perturbations to the tissue microenvironment (stiffening, inflammation, edema causing osmotic stress), lead to age-dependent cellular dysfunction by altering the dynamics of assembly, disassembly and transport of macromolecular protein machines. We will test this hypothesis in cellular models, animal models, and patient tissue by (1) using novel tools to probe the intracellular biophysical environment of cells; (2) integrating these findings using novel genomics technologies applied to mouse models to study (i) how intracellular changes in crowding and extracellular changes in the tissue microenvironment may drive pathogenesis in vivo, and (ii) how such perturbations disrupt cell-cell communication in vulnerable regions of tissue; and (3) relating our findings to human disease by re-examining these findings in the context of a clinically and neuropathologically deeply curated cohort of ALS/FTD patients. These studies will allow us to address the following questions: 1) Why does abnormal protein aggregation and accumulation occur in sporadic disease, and how might this contribute to disease pathogenesis; 2) Do these alterations in protein homeostasis perturb intercellular communication in the tissue microenvironment; and 3) What drives the cell type vulnerability that makes ALS/FTD unique? The proposed work will accomplish the following: A) represent the first detailed survey of molecular crowding in neural cells; B) uncover whether a causal link between intracellular crowding, protein aggregation, and neurodegeneration exists, C) establish whether the impacts of intracellular crowding show cell type specific signatures including changes in protein- protein interactions, and D) provide a new framework to explore therapeutic strategies for treating ALS/FTD.