Defining cell type-specific functions for the selective autophagy receptor p62 in neurons and astrocytes

定义神经元和星形胶质细胞中选择性自噬受体 p62 的细胞类型特异性功能

• 批准号: 10676686
• 负责人: DAVID Kader SIDIBE
• 金额: $ 4.77万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10676686
• 关键词: Affect; Antioxidants; Area; Astrocytes; Autophagocytosis; Autophagosome; Binding; Cells; Cellular Stress; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Cytoplasm; Data; Defect; Degradation Pathway; Disease; Genes; Goals; Impairment; Investigation; Knowledge; Link; Lysosomes; Measures; Metabolic stress; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Oxidative Stress; Oxidative Stress Induction; Pathology; Pathway interactions; Proteins; Proteome; Regulation; Resolution; Role; Serine; Small Interfering RNA; Specific qualifier value; Stress; Structure; System; Testing; Ubiquitin; Ubiquitination; Western Blotting; bafilomycin A1; biological adaptation to stress; cell type; combat; design; falls; in vivo; inhibitor; insight; knock-down; mutant; neuroprotection; protein aggregation; proteotoxicity; receptor; response; superoxide dismutase 1; targeted treatment; therapeutic development; transcription factor

Project Summary. Neurons and astrocytes have unique demands in regulating the quality of their proteome. A key regulator of the proteome is autophagy, a lysosomal degradation pathway. During autophagy, cytoplasmic components are packaged into autophagosomes and delivered to lysosomes for cargo degradation. Autophagy is neuroprotective, as mutations in key autophagy genes cause neurodegeneration. Preliminary studies show that autophagy is regulated differently in neurons and astrocytes in multiple paradigms of stress. Despite the importance of autophagy, how it is regulated in neurons and astrocytes to facilitate cell- type specific functions and stress responses is largely unknown. Thus, the goal for this proposal is to define cell-type specific functions for the autophagy receptor p62 in neurons and astrocytes. P62 facilitates selective forms of autophagy by binding to ubiquitinated substrates and the autophagy marker LC3, thereby incorporating cargo into a forming autophagosome. P62 mitigates proteotoxic stress by targeting protein aggregates to the autophagy pathway. Additionally, p62 mitigates oxidative stress by targeting Keap1, a negative regulator of the antioxidant transcription factor NRF2, for degradation by autophagy. To examine functions of p62 in each cell type, we established a robust system to coculture neuron and astrocytes. This system recapitulates intercellular interactions found in vivo, and provides an easily manipulatable system for studying cell-type specific p62 function with high resolution. Using the coculture, I found by immunostain that metabolic stress (autophagy activator) induces formation of p62-ubiquitin positive structures (i.e., ubiquitinated cargo) only in neurons. Moreover, blocking ubiquitination significantly reduces p62 puncta formation and degradation in neurons as compared to astrocytes in basal and stress conditions. Astrocytes are crucial to combating oxidative stress, but the role of p62 in this pathway in astrocytes is largely unknown. I found that oxidative stress induces p62 levels selectively in astrocytes. Thus, I hypothesize that p62 functions primarily in selective autophagy in neurons, and primarily in the antioxidant pathway in astrocytes. Importantly, ALS-linked mutations in p62 fall in domains important for each function. But how p62 protects against neurodegeneration in each cell type is not understood. I hypothesize that ALS-linked mutations in p62 domains that are important for selective autophagy and antioxidant function will impair p62 function in neurons and astrocytes, respectively. To test these hypotheses, I will (Aim 1) define cell-type specific functions of p62 in neurons and astrocytes, and (Aim 2) determine the effects of ALS-linked mutations on p62 function in neurons and astrocytes. This study will elucidate cell-type specific contributions of neurons and astrocytes to neurodegeneration. In turn, understanding cell-type specific contributions to ALS will enable opportunities for more targeted and specified therapies to mitigate neurodegeneration.

项目摘要。神经元和星形胶质细胞在调节其蛋白质组的质量方面具有独特的需求。一 蛋白质组的关键调节因子是自噬，一种溶酶体降解途径。在自噬过程中， 将这些组分包装到自噬体中并递送到溶酶体用于货物降解。 自噬具有神经保护作用，因为关键自噬基因的突变会导致神经退行性变。初步 研究表明，在多种应激模式中，神经元和星形胶质细胞中的自噬受到不同的调节。 尽管自噬的重要性，它是如何在神经元和星形胶质细胞中调节，以促进细胞- 类型特异性功能和应激反应在很大程度上是未知的。因此，本提案的目标是 定义神经元和星形胶质细胞中自噬受体p62的细胞类型特异性功能。P62有助于 通过结合泛素化底物和自噬标记物LC 3的选择性自噬形式，从而 将货物并入形成的自噬体中。P62通过靶向蛋白减轻蛋白毒性应激 自噬途径的聚集体。此外，p62通过靶向Keap 1减轻氧化应激， 抗氧化转录因子NRF 2的负调节因子，用于通过自噬降解。审查 p62在每种细胞类型中的功能，我们建立了一个强大的系统来共培养神经元和星形胶质细胞。这 系统概括了体内发现的细胞间相互作用，并提供了一种易于操作的系统， 高分辨率研究细胞类型特异性p62功能。通过免疫染色，我发现， 代谢应激（自噬激活剂）诱导p62-遍在蛋白阳性结构的形成（即，泛素化 货物）仅在神经元中。此外，阻断泛素化可显著减少p62斑点的形成， 与基础和应激条件下的星形胶质细胞相比，神经元中的降解。星形胶质细胞对于 抗氧化应激，但在星形胶质细胞中p62在这一途径中的作用在很大程度上是未知的。我发现 氧化应激选择性诱导星形胶质细胞中的p62水平。因此，我假设p62功能 主要在神经元中的选择性自噬中，以及主要在星形胶质细胞中的抗氧化剂途径中。 重要的是，ALS连锁突变p62落在域重要的每一个功能。P62如何保护 对每种细胞类型的神经变性的影响尚不清楚。我假设ALS相关突变 对于选择性自噬和抗氧化功能重要的p62结构域将损害p62 分别在神经元和星形胶质细胞中发挥作用。为了验证这些假设，我将（目标1）定义细胞类型 p62在神经元和星形胶质细胞中的特异性功能，并（目的2）确定ALS连锁突变的影响 p62在神经元和星形胶质细胞中的功能。这项研究将阐明神经元的细胞类型特异性贡献 和星形胶质细胞转化为神经变性。反过来，了解细胞类型对ALS的特异性贡献将使 更多的针对性和特定的治疗，以减轻神经退行性变的机会。