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

• 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
• 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' 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

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激活因子动力蛋白的突变可导致神经性疾病，包括遗传性疾病 运动神经元变性和Charcot Marie Tooth病的形式。更广泛地说，我们知道 动力蛋白驱动的功能，如逆行轴突运输，参与了糖尿病的发病机制。 神经退行性疾病，包括肌萎缩侧索硬化症(ALS)、亨廷顿氏症和阿尔茨海默氏症。 然而，具体涉及的机制仍不清楚。动力蛋白是一种多效性细胞马达，具有多个 在神经元中扮演不同的角色。在这里，我们将重点研究动力蛋白驱动的逆行中的缺陷这一假设 包括溶酶体和自噬在内的降解细胞器的运输是导致 以这些疾病为特征的轴突变性。这项建议的目标是了解具体的 将动力蛋白功能缺陷与神经退行性变联系起来的机制主要集中在以下三个目标：(1) 在神经退行性变过程中，逆行轴突运输是如何改变的？我们假设病态的 JNK和CDK5通路的改变导致对立的微管马达在 轴突运输。我们将使用囊泡运输的定量活细胞成像来验证这一假设 来自多种ALS模型的原代神经元。然后，我们将机械地剖析激酶是如何错误调节的 使用单分子分辨率的体外重建方法影响运动功能。这些研究 将测试模型，即方向相反的电机协调中断是主要缺陷 导致沿着轴突的运输发生改变。(2)自噬小体生物发生的途径有哪些 在神经元中装载货物？我们假设神经元中的自噬遵循刻板印象 维持细胞内稳态所需的空间调节途径。我们将检查自噬小体 利用定量活细胞成像技术在初级感觉神经元和运动神经元中的生物发生和载物 重点研究动力蛋白和视神经磷酸酶的作用。然后我们将确定这一途径如何响应细胞 应激源，以解决这一途径对细胞反应的上调能力有限的假设 压力。(3)动力蛋白驱动的自噬缺陷是如何导致轴突退化的？我们 假设主动的、动力蛋白驱动的自噬小体的运输与功能密切相关，并且 运输过程中的缺陷会导致老化细胞器和聚集蛋白的缺陷降解。我们将使用 用活体成像、生化和细胞分析来确定自噬小体中的缺陷如何运输 轴突导致神经变性以及不同的动力蛋白突变是如何不同地扰乱细胞的 功能，导致不同的临床表现。细胞质动力蛋白的突变足以导致 人类神经退行性疾病包括脊髓性肌萎缩症(SMA-LED)和Charcot-Marie-Tooth 疾病(2O型)，但涉及的机制仍有待确定。在实现这些目标方面应取得进展 为治疗方法或临床干预提供新的机会。

项目成果

What Doesn't Kill You Makes You Stronger.

那些杀不死你的会让你变得更强大。

• DOI: 10.1016/j.devcel.2018.11.003
• 发表时间: 2018
• 期刊: Developmental cell
• 影响因子: 11.8
• 作者: Stavoe,AndreaKH;Holzbaur,ErikaLF
• 通讯作者: Holzbaur,ErikaLF

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.

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

• DOI: 10.1016/j.neulet.2018.03.025
• 发表时间: 2019-04-01
• 期刊: Neuroscience letters
• 影响因子: 2.5
• 作者: Stavoe AKH;Holzbaur ELF
• 通讯作者: Holzbaur ELF

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

Neuroligin 1 is dynamically exchanged at postsynaptic sites.

• DOI: 10.1523/jneurosci.0896-10.2010
• 发表时间: 2010-09-22
• 期刊: The Journal of neuroscience : the official journal of the Society for Neuroscience
• 作者: Schapitz IU;Behrend B;Pechmann Y;Lappe-Siefke C;Kneussel SJ;Wallace KE;Stempel AV;Buck F;Grant SG;Schweizer M;Schmitz D;Schwarz JR;Holzbaur EL;Kneussel M
• 通讯作者: Kneussel M