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

TDP-43 神经病理学中生物分子凝聚物形成的分子驱动因素的体内表征

基本信息

批准号：
10698165
负责人：
Marco Morsch
金额：
$ 14.51万
依托单位：
MACQUARIE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-15 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10698165
关键词：
ALS patients Acceleration Affect Age Aged, 80 and over Alzheimer&apos s Disease Amino Acid Substitution Amyotrophic Lateral Sclerosis Binding Biological Models C-terminal Cell Nucleus Cell physiology Cells Characteristics Chromatin Cytoplasm DNA Dementia Development Disease Disease Progression Etiology Fishes Fluorescence Recovery After Photobleaching Frontotemporal Lobar Degenerations Future Genes Glycine Homeostasis Imaging Device Imaging Techniques In Vitro Individual Knowledge Liquid substance Messenger RNA Microscopy Modeling Molecular Monitor Motor Neuron Disease Motor Neurons Mutation Nerve Degeneration Neurodegenerative Disorders Neurons Nuclear Nuclear Protein Outcome Parkinson Disease Pathogenesis Pathologic Pathology Persons Phase Phase Transition Physical condensation Physiological Positioning Attribute Post-Translational Protein Processing Process Property Protein Deficiency Proteins RNA Binding RNA Recognition Motif RNA Splicing RNA Transport RNA-Binding Proteins Reporter Resolution Role Spinal Cord Stress TDP-43 aggregation Techniques Technology Text Therapeutic Intervention Time Transcript Translations Variant Zebrafish collaborative approach cost effective therapy familial amyotrophic lateral sclerosis in vivo in vivo imaging insight limbic-predominant age-related TDP-43 encephalopathy molecular imaging mutant nanoscale neuropathology new technology novel novel therapeutics nucleocytoplasmic transport optogenetics protein TDP-43 protein aggregation quantitative imaging single molecule superresolution microscopy tool

项目摘要

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.

项目摘要/摘要（文本30行）肌萎缩性外侧硬化症（ALS）是一种运动神经元疾病（MND），其特征是特异性变性上和/或下运动神经元的。关键神经病理学，一种称为蛋白质的不溶骨料 TDP-43是几乎所有（约97％）ALS患者的证据。 TDP-43骨料也存在于其他几个神经退行性疾病，包括阿尔茨海默氏病（AD），痴呆症（FTLD）和边缘疾病与年龄有关的TDP-43脑病（晚期；大约25％的80岁以上的人发现）。 TDP-43是一种普遍存在的核蛋白，由TARDBP基因编码，并且在极少数ALS-FTLD或FTLD的情况下，突变是家庭ALS的原因。 TDP-43是RNA结合通过许多细胞调节来调节自身和数千个mRNA转录本的蛋白质过程（例如剪接，RNA传输，翻译等）。 TDP-43聚集可能是发病机理期间的收敛点，目的地可能有可能不同的病因和上游机制。但是，确定起源和机制与神经变性相关的蛋白质聚集的贡献被证明是一个主要挑战田地（Goedert and Spillantini 2006）。这是这项研究的总体目的是更好地了解驱动生物分子冷凝物（BMC）形成的细胞和分子机制单元，实时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，因此影响了在神经退行性疾病。确定驱动BMC形成，蛋白质缺陷和病理对于发展新型治疗途径至关重要。体外证据表明，TDP-43经历液体去混合（液 - 液相分离， LLP）进入BMC，以调节其在神经元内的物理水平和定位（Tziortzouda，van den Bosch等。 2021）。但是，在体内证据仍然缺乏。在此提案中，我们提供 BMC表征的初步证据在活斑马鱼的脊髓中。我们建议使用一套高级分子成像工具，包括光漂白后的荧光回收（FRAP），单分子跟踪（SMT）和光遗传学结合超分辨率显微镜方法（DSTORM）。我们将评估翻译后如何修饰（PTM）和单氨基酸取代会影响BMC特性，并导致病理学 TDP-43体内TDP-43的错误定位（Buratti 2018）。综上所述，该建议将大大加速监测BMC形成的可能性通过在我们既定的斑马鱼平台上实施新技术，并提供急需了解BMC形成如何影响神经退行性疾病范围的关键病理。