Mechanistic analysis of axonal transport defects in neurodegenerative disease

神经退行性疾病轴突运输缺陷的机制分析

基本信息

批准号：
9896888
负责人：
Erika L Holzbaur
金额：
$ 45.83万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-04-01 至 2021-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9896888
关键词：
ALS patients Address Affect Afferent Neurons Aging Alzheimer&apos s Disease Amyotrophic Lateral Sclerosis Autophagocytosis Autophagosome Axon Axonal Transport Back Biochemical Biogenesis Cell physiology Cellular Assay Cellular Stress Charcot-Marie-Tooth Disease Clinical Data Defect Degenerative Disorder Degradation Pathway Disease Dynein ATPase Goals Heritability Homeostasis Human Huntington Disease Image In Vitro Intervention Intracellular Transport Kinesin Lead Length Link Lysosomes MAPK8 gene Mediating Microtubules Modeling Molecular Motors Motor Motor Neurons Movement Mutation Nerve Degeneration Neurodegenerative Disorders Neurons Organelles Pathogenicity Pathologic Pathology Pathway interactions Phosphotransferases Proteins Regulation Regulatory Pathway Resolution Role Spinal Muscular Atrophy Testing Therapeutic Vesicle axonal degeneration dynactin experimental study gene therapy genetic analysis insight live cell imaging meter motor neuron degeneration mouse model neuron loss protein aggregation reconstitution response retrograde transport single molecule stressor vesicle transport

项目摘要

Project Summary Mutations in cytoplasmic dynein or its activator dynactin are causative for neuronal diseases including heritable forms of motor neuron degeneration and CharcotMarieTooth disease. More broadly, we know that defects in dyneindriven functions such as retrograde axonal transport are involved in the pathogenic mechanisms of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), Huntington’s, and Alzheimer’s. However, the specific mechanisms involved remain unclear. Dynein is a pleiotropic cellular motor with multiple distinct roles in the neuron. Here we will focus on the hypothesis that defects in the dyneindriven retrograde transport of degradative organelles including lysosomes and autophagosomes are major contributors to the axonal degeneration that characterize these diseases. The goal of this proposal is to understand the specific mechanisms linking defects in dynein function to neurodegeneration, focusing on the following three aims: (1) How is retrograde axonal transport altered during neurodegeneration? We hypothesize that pathological alterations in the JNK and Cdk5 pathways lead to the dysregulation of opposing microtubule motors during axonal transport. We will test this hypothesis using quantitative live cell imaging of vesicular transport in primary neurons from multiple models of ALS. Then, we will mechanistically dissect how kinase misregulation affects motor function using in vitro reconstitution approaches with single molecule resolution. These studies will test the model that a disruption in the coordination of oppositelyoriented motors is the primary defect leading to altered transport along the axon. (2) What are the pathways for autophagosome biogenesis and cargoloading in the neuron? We hypothesize that autophagy in the neuron follows a stereotypical and spatially regulated pathway that is required to maintain cellular homeostasis. We will examine autophagosome biogenesis and cargoloading in primary sensory and motor neurons using quantitative live cell imaging, focusing on the roles of dynein and optineurin. Then we will determine how this pathway responds to cellular stressors, to address the hypothesis that this pathway has a limited ability to upregulate in response to cellular stress. (3) How do defects in dyneindriven autophagy lead to degeneration of the axon? We hypothesize that the active, dyneindriven transport of autophagosomes is tightly linked to function, and that defects in transport will lead to defective degradation of aging organelles and aggregated proteins. We will use live imaging and biochemical and cellular assays to determine how defects in autophagosome transport along the axon contribute to neurodegeneration and how distinct dynein mutations differentially perturb cellular functions, leading to disparate clinical manifestations. Mutations in cytoplasmic dynein are sufficient to cause human neurodegenerative diseases including spinal muscular atrophy (SMALED) and CharcotMarieTooth disease (Type 2O), but the mechanisms involved remain to be determined. Progress on these aims should offer new opportunities for therapeutic approaches or clinical intervention.

项目摘要细胞质动力蛋白或其活化剂dynactin的突变对于神经元疾病（包括遗传）是严重的运动神经元变性的形式和熟食疾病。更广泛地，我们知道缺陷大道鉴定功能（例如逆行轴突运输）参与了致病机制神经退行性疾病，包括肌萎缩性侧面硬化症（ALS），亨廷顿和阿尔茨海默氏症。但是，涉及的具体机制尚不清楚。动力蛋白是一种多效细胞电动机在神经元中的独特作用。在这里，我们将重点介绍Dyneindriven逆行缺陷的假设降解细胞器的运输在内这些疾病的特征是轴突变性。该提议的目的是了解特定的将动力蛋白功能缺陷与神经变性联系起来的机制，重点是以下三个目的：（1）神经变性过程中逆行轴突转运如何改变？我们假设这种病理 JNK和CDK5途径的改变导致相反的微管电机的失调轴突运输。我们将使用囊泡转运的定量活细胞成像检验该假设来自多种ALS的主要神经元。然后，我们将机械地剖析激酶不调节使用单分子分辨率的体外重构方法影响运动功能。这些研究将测试模型，即对相反电动机的协调造成的破坏是主要缺陷导致沿轴突的转运改变。（2）自噬体生物发生的途径和神经中的碳脂加载？我们假设神经元中的自噬遵循刻板印象和维持细胞稳态所需的空间调节途径。我们将检查自噬体使用定量的活细胞成像中的原发性感觉和运动神经元中的生物发生和碳脂加载，专注于动力蛋白和果蛋白神经素的作用。然后，我们将确定该途径如何响应细胞应力源，以解决以下假设：该途径对细胞的上调能力有限压力。（3）Dyneindriven自噬的缺陷如何导致轴突退化？我们假设自噬体的活性，大动症的运输与功能紧密相关，并且运输缺陷将导致衰老细胞器和聚集蛋白的降解缺陷。我们将使用实时成像以及生化和细胞测定法，以确定自噬体中的缺陷如何沿着轴突有助于神经变性以及不同的动力蛋白突变如何不同地扰动细胞功能，导致不同的临床表现。细胞质动力蛋白的突变足以引起人类神经退行性疾病，包括脊柱肌肉萎缩（SMALED）和Charcotmarietooth 疾病（2O型），但涉及的机制仍有待确定。这些目标的进步应该为治疗方法或临床干预提供新的机会。

项目成果

期刊论文数量（27）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Vesicular degradation pathways in neurons: at the crossroads of autophagy and endo-lysosomal degradation

DOI：
10.1016/j.conb.2019.01.005
发表时间：
2019-08-01
期刊：
CURRENT OPINION IN NEUROBIOLOGY
影响因子：
5.7
作者：
Boecker, C. Alexander;Holzbaur, Erika L. F.
通讯作者：
Holzbaur, Erika L. F.

ALS- and FTD-associated missense mutations in TBK1 differentially disrupt mitophagy.

DOI：
10.1073/pnas.2025053118
发表时间：
2021-06-15
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Harding O;Evans CS;Ye J;Cheung J;Maniatis T;Holzbaur ELF
通讯作者：
Holzbaur ELF

Axonal autophagy: Mini-review for autophagy in the CNS.