Dissecting the role of NEU1-dependent de-sialylation in neurodegeneration and neuroinflammation

剖析 NEU1 依赖性去唾液酸化在神经退行性变和神经炎症中的作用

• 批准号: 10279649
• 负责人: ALESSANDRA D'AZZO
• 金额: $ 175.06万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-15 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10279649
• 关键词: Adult; Affect; Alzheimer' s Disease; Alzheimer' s disease model; Amyloid; Amyloid beta-Protein Precursor; Amyloid deposition; Amyloidosis; Anti-Inflammatory Agents; Applications Grants; Biochemical; Biological Process; Brain; Carboxypeptidase; Catabolism; Cell Fractionation; Cell membrane; Cell physiology; Cells; Characteristics; Charge; Childhood; Cleaved cell; Complex; Data; Deposition; Disease; Enzymes; Event; Exocytosis; Experimental Designs; Functional disorder; Genetic; Glycoconjugates; Glycoproteins; Hippocampus (Brain); Homeostasis; Human; Image; Impairment; In Vitro; Individual; Knowledge; Laboratories; Lead; Link; Lipomucopolysaccharidoses; Lysosomal Storage Diseases; Lysosomes; Mammalian Cell; Mediating; Membrane Proteins; Methods; Microglia; Microscopy; Modeling; Molecular; Monosaccharides; Morphology; Mus; Nerve Degeneration; Neuraminidase; Neurodegenerative Disorders; Neurons; Parkinson Disease; Pathogenesis; Pathogenicity; Pathologic Processes; Patients; Peptide Hydrolases; Phagocytes; Phagocytosis; Pharmacology; Phenotype; Physiological; Physiological Processes; Polysaccharides; Positioning Attribute; Post-Translational Protein Processing; Process; Property; Proteins; Proteolytic Processing; Receptor Signaling; Reporting; Resolution; Role; Sialic Acids; Sialoglycoproteins; Signaling Molecule; Signaling Protein; TREM2 gene; Techniques; Testing; Therapeutic; Three-Dimensional Imaging; Tissues; Work; amyloid formation; beta secretase; beta-site APP cleaving enzyme 1; carboxypeptidase C; cell type; direct application; experimental study; extracellular; gamma secretase; gene therapy; insight; mouse model; neuroinflammation; neuropathology; nicastrin protein; novel therapeutic intervention; peptide B; public health relevance; receptor; relating to nervous system; sialylation; sugar; tool

ABSTRACT Changes in the sialic acid content of neural glycoproteins have been reported in neurodegenerative and neuroinflammatory diseases, including Alzheimer’s disease (AD). However, how these changes govern neuropathogenic processes remains poorly understood. The central hypothesis of these studies is that impaired de-sialylation affects the physiological and functional properties of specific sialo-glycoproteins in neurons and microglia, initiating a pathogenic cascade leading to neurodegeneration and neuroinflammation. We will test this hypothesis from the angle of the sialic acid-cleaving enzyme NEU1, a lysosomal sialidase, using a mouse model of the lysosomal storage disease, sialidosis, as an experimental tool. We have shown that Neu1–/– mice develop a hippocampal neuropathology with features of an amyloidosis, including accumulation of an oversialylated APP, a substrate of NEU1, and widespread neuroinflammation. Ablating NEU1 expression in a canonical model of AD (5xFAD) leads to exacerbation of the amyloidosis phenotype, while increasing NEU1 expression, via a gene therapy approach, reduces plaque formation. These findings implicate NEU1 as the potential underpinning cause, although the mechanisms linking the cause to the effects still need to be deciphered. The studies proposed in this application are directed to closing this knowledge gap, because of these previous observations and new compelling preliminary data. Besides APP, we have identified additional potential substrates of NEU1, including those that participate directly in the amyloidogenic process (BACE1 and nicastrin) and those that are responsible for neuroinflammatory processes, such as microglial phagocytosis (CD68 and TREM2). We will explore the idea that by controlling the levels of sialic acids on these specific AD-related glycoproteins in neurons (Aim 1) and microglia (Aim 2), NEU1 functions as a crucial regulator of key biological processes that maintain brain homeostasis. In Aim 1, we will examine the effects of altered sialylation on APP, and its sialylated proteolytic enzymes, BACE1 and nicastrin, in Neu1–/– mice and in established AD mouse models crossed into the Neu1–/– background. We will use a combination of subcellular fractionation methods, immunofluorescent microscopy, and high-resolution 3D imaging to determine alterations in intracellular distribution, processing and turnover rate of these glycoproteins. In Aim2, we will study how impaired de-sialylation of microglial receptors and signaling proteins affects their subcellular localization, turnover rate, and the cellular functions they are known to control. To focus our studies exclusively on microglia we will cross the above-mentioned mice with a mouse model that expresses GFP almost solely in microglia. Overall the experimental design of this grant application will enable us to elucidate mechanisms of pathogenesis leading to neuronal and microglial dysfunction with direct translational implications for patients with AD.

摘要 神经糖蛋白中唾液酸含量的变化已经在神经退行性疾病和神经退行性疾病中被报道。 神经炎性疾病，包括阿尔茨海默病（AD）。然而，这些变化如何管理 神经病理过程仍然知之甚少。这些研究的中心假设是， 受损的去唾液酸化作用影响特定唾液酸糖蛋白的生理和功能特性， 神经元和小胶质细胞，启动导致神经变性和神经炎症的致病级联反应。 我们将从唾液酸裂解酶NEU 1（一种溶酶体唾液酸酶）的角度来检验这一假设， 使用溶酶体贮积病，唾液酸沉积症的小鼠模型作为实验工具。我们已经表明 Neu 1-/-小鼠出现具有淀粉样变性特征的海马神经病理学，包括 过度唾液酸化APP（NEU 1的底物）的积累和广泛的神经炎症。消融 在AD（5xFAD）的典型模型中NEU 1表达导致淀粉样变性表型的恶化， 而通过基因治疗方法增加NEU 1的表达减少了斑块的形成。这些发现 暗示NEU 1是潜在的基础原因，尽管将原因与结果联系起来的机制 仍然需要被破译。本申请中提出的研究旨在关闭这些知识 差距，因为这些以前的意见和新的令人信服的初步数据。除了APP，我们还有 鉴定了NEU 1的其他潜在底物，包括直接参与淀粉样蛋白生成的那些底物。 过程（BACE 1和nicastrin）和那些负责神经炎症过程，如 小胶质细胞吞噬作用（CD 68和TREM 2）。我们将探讨通过控制唾液酸水平 在神经元（Aim 1）和小胶质细胞（Aim 2）中，NEU 1的功能是 是维持大脑稳态的关键生物过程的重要调节器。在目标1中，我们将检查 Neu 1-/-中唾液酸化改变对APP及其唾液酸化蛋白水解酶BACE 1和nicastrin的影响 小鼠和已建立的AD小鼠模型中，与Neu 1-/-背景杂交。我们将使用一个组合 亚细胞分离方法，免疫荧光显微镜和高分辨率3D成像， 确定这些糖蛋白的细胞内分布、加工和周转率的改变。在目标2中， 我们将研究小胶质细胞受体和信号蛋白的去唾液酸化受损如何影响它们的亚细胞 定位，周转率，以及已知它们控制的细胞功能。把我们的研究集中在 在小胶质细胞上，我们将上述小鼠与几乎仅表达GFP的小鼠模型杂交， 在小胶质细胞中。总的来说，这项资助申请的实验设计将使我们能够阐明机制 导致神经元和小胶质细胞功能障碍的发病机制， AD患者

项目成果

Glycosphingolipids within membrane contact sites influence their function as signaling hubs in neurodegenerative diseases.

• DOI: 10.1002/2211-5463.13605
• 发表时间: 2023-09
• 期刊: FEBS OPEN BIO
• 影响因子: 2.6
• 作者: Weesner, Jason Andrew;Annunziata, Ida;van de Vlekkert, Diantha;d'Azzo, Alessandra
• 通讯作者: d'Azzo, Alessandra