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（30 行文本）肌萎缩侧索硬化症 (ALS) 是一种运动神经元疾病 (MND)，其特征为特异性变性上运动神经元和/或下运动神经元的关键神经病理学是一种称为不溶性蛋白质的聚集体。 TDP-43 在几乎所有 (~97%) ALS 患者中都很明显，TDP-43 聚集体也存在于其他几种患者中。神经退行性疾病，包括阿尔茨海默病 (AD)、痴呆 (FTLD) 和边缘主导型痴呆年龄相关的 TDP-43 脑病（晚期；约 25% 的 80 岁以上个体中发现）。 TDP-43 是一种普遍存在的主要核蛋白，由 TARDBP 基因编码，突变是家族性 ALS 的一个原因，在极少数情况下，ALS-FTLD 或 FTLD 是 RNA 结合。通过调节许多细胞来调节自身和数千个 mRNA 转录物的蛋白质过程（例如剪接、RNA 运输、翻译等）。 TDP-43 聚集很可能作为发病机制中的汇聚点，尽管有可能不同的病因和上游机制然而，确定其起源和机制。促进与神经退行性变相关的蛋白质聚集被证明是该领域的一个主要挑战因此，本研究的首要目的是更好地了解驱动生物体中生物分子凝聚体 (BMC) 形成的细胞和分子机制我们将使用我们的斑马鱼模型系统实时研究基本的分子过程。调节 TDP-43 相分离，从而影响受到影响的细胞稳态确定驱动 BMC 形成、蛋白质缺乏和的过程。病理学对于开发新的治疗途径至关重要。体外证据表明 TDP-43 经历流体分层（液-液相分离， LLPS）进入 BMC 以调节其在神经元内的生理水平和定位（Tziortzouda，Van Den Bosch 等人，2021）但是，我们在该提案中仍然缺乏该过程的体内证据。我们建议活体斑马鱼脊髓中 BMC 特征的初步证据。使用一套先进的分子成像工具进一步表征我们的鱼的这一过程，包括光漂白后的荧光恢复 (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，我们将评估翻译后的效果。修饰 (PTM) 和单一氨基酸取代会影响 BMC 特性并导致病理性 TDP-43 在体内的错误定位 (Buratti 2018)。总而言之，该提案将显着加快监测 BMC 形成的可能性 vivo 通过在我们建立的斑马鱼平台上实施新技术，并提供急需的深入了解 BMC 的形成如何影响一系列神经退行性疾病的关键病理学。