In vivo characterization of the molecular drivers of biomolecular condensate formation in TDP-43 neuropathology

项目摘要/摘要（30行文本） 肌萎缩侧索硬化症是一种以特异性变性为特征的运动神经元疾病 上和/或下运动神经元。关键的神经病理学，一种叫做 TDP-43在几乎所有（~97%）ALS患者中均明显。TDP-43聚集体也存在于几种其他的细胞中。 神经退行性疾病，包括阿尔茨海默病（AD）、痴呆（FTLD）和边缘系统主导型痴呆（BAD）。 年龄相关的TDP-43脑病（LATE;在80岁以上的个体中发现约25%）。 TDP-43是由TARDBP基因编码的普遍存在的且占主导地位的核蛋白， 突变是家族性ALS的原因，在ALS-FTLD或FTLD的罕见病例中。TDP-43是一种RNA结合蛋白， 一种蛋白质，通过调节许多细胞因子来调节自身和数千种mRNA转录物。 过程（例如剪接、RNA转运、翻译等）。 TDP-43聚集可能在发病过程中充当汇聚点，尽管可能存在潜在的 不同的病因和上游机制。然而，确定的起源和机制， 促进与神经变性相关的蛋白质聚集被证明是神经变性研究中的一个主要挑战。 field（Goedert and Spillantini 2006）.因此，本研究的总体目标是更好地了解 细胞和分子机制，驱动生物分子凝聚物（BMC）形成的生活 细胞，在真实的时间，使用我们的斑马鱼模型系统。我们将研究基本的分子过程 调节TDP-43相分离，从而影响细胞内稳态， 神经退行性疾病确定驱动BMC形成、蛋白质缺乏和 病理学对于开发新的治疗途径是必要的。 体外证据表明TDP-43经历流体分层（液-液相分离， LLPS）进入BMC以调节其生理水平和在神经元内的定位（Tziortzouda，货车Den Bosch等人，2021年）。然而，这一过程的体内证据仍然缺乏。在本提案中，我们提供 活体斑马鱼脊髓中BMC特征的初步证据。我们建议 使用一套先进的分子成像工具进一步描述我们鱼类的这一过程，包括 光漂白后荧光恢复（FRAP）、单分子追踪（SMT）和光遗传学 与超分辨率显微镜方法（dSTORM）相结合。我们将评估翻译后 修饰（PTM）和单个氨基酸取代可以影响BMC性质并导致病理性的 TDP-43在体内的错误定位（Buratti 2018）。 总之，这一建议将大大加快监测BMC形成的可能性， 通过在我们建立的斑马鱼平台上实施新技术， 了解BMC形成如何影响神经退行性疾病谱中的关键病理学。

Project Summary/Abstract (30 lines of text) Amyotrophic lateral sclerosis (ALS) is a motor neuron disease (MND) characterized by specific degeneration of upper and/or lower motor neurons. The key neuropathology, insoluble aggregates of a protein called TDP-43, is evident in almost all (~97%) ALS patients. TDP-43 aggregates are also present in several other neurodegenerative diseases, including Alzheimer’s disease (AD), dementias (FTLD), and limbic predominant age-related TDP-43 encephalopathy (LATE; found in about 25% of individuals over the age of 80). TDP-43 is a ubiquitous and predominately nuclear protein that is encoded by the TARDBP gene and mutations are a cause of familial ALS, in rare cases of ALS-FTLD or FTLD. TDP-43 is an RNA binding protein that regulates itself and thousands of mRNA transcripts via the modulation of numerous cellular processes (e.g. splicing, RNA transport, translation and more). TDP-43 aggregation is likely to serve as a convergence point during pathogenesis, despite potentially different etiologies and upstream mechanisms. However, identifying the origins and mechanisms that contribute to protein aggregation associated with neurodegeneration proves to be a major challenge in the field (Goedert and Spillantini 2006). Thus, the overarching aim of this study is to better understand the cellular and molecular mechanisms that drive biomolecular condensate (BMC) formation in a living cell, in real time, using our zebrafish model system. We will investigate fundamental molecular processes that regulate TDP-43 phase separation and therefore influence the cellular homeostasis that is affected in neurodegenerative diseases. Determining the processes that drive BMC formation, protein deficiencies, and pathology are imperative for the development of novel therapeutic avenues. In vitro evidence indicates that TDP-43 undergoes fluid de-mixing (liquid-liquid phase separation, LLPS) into BMCs to regulate its physiological levels and localization within a neuron (Tziortzouda, Van Den Bosch et al. 2021). However, in vivo evidence of this process is still lacking. In this proposal, we provide preliminary evidence of BMC characterization in the spinal cord of living zebrafish. We propose to further characterize this process in our fish using a suite of advanced molecular imaging tools, including Fluorescence Recovery After photobleaching (FRAP), Single Molecule Tracking (SMT), and optogenetics in combination with super-resolution microscopy approaches (dSTORM). We will assess how posttranslational modifications (PTMs) and single amino acid substitutions can affect BMC properties and lead to pathological mislocalization of TDP-43 in vivo (Buratti 2018). Taken together, this proposal will significantly accelerate the possibilities to monitor BMC formation in vivo by implementing novel technologies in our established zebrafish platform, and provide much needed insight on how BMC formation can affect a key pathology in a spectrum of neurodegenerative diseases.