BMP-dependent pathways and Alzheimer's disease

BMP 依赖性途径与阿尔茨海默氏病

基本信息

批准号：
10511117
负责人：
Catherine A Marquer
金额：
$ 16.45万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
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 2023-03-20
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10511117
关键词：
Address Adult Affect Age Alzheimer&apos s Disease Alzheimer&apos s disease pathology Alzheimer&apos s disease patient Amyloid Anabolism Antibodies Astrocytes Autopsy Biological Biological Assay Brain Brain region Cell Culture Techniques Cell Line Cells Cerebellum Cholesterol Confocal Microscopy Critical Pathways Data Dementia with Lewy Bodies Disease Disease Progression Down Syndrome Ensure Enzymes Eukaryotic Cell Fibroblasts Freezing Future Gender Gene Expression Gene Proteins Grant Hela Cells Hippocampus (Brain)Homeostasis Human In Vitro Individual Intracellular Transport Late Onset Alzheimer Disease Link Lipids Liposomes Literature Lysosomes Membrane Microglia Multivesicular Body Mus Nerve Degeneration Neurodegenerative Disorders Neuronal Ceroid-Lipofuscinosis Neurons New York Organelles Outcome Pathogenesis Pathologic Pathology Pathway interactions Patients Phosphatidylinositols Phospholipase A2 Physiological Prefrontal Cortex Production Proteins Recycling Reporting Role Route Sampling Sorting - Cell Movement Stains Testing United States National Institutes of Health Vesicle Western Blotting Wild Type Mouse bis(monoacylglyceryl)phosphate brain cell brain health cell type entorhinal cortex exosome extracellular late endosome lipidomics mouse model novel therapeutics overexpression protein expression transcriptome sequencing

项目摘要

SUMMARY Amongst lipids that regulate intracellular transport routes, bis(monoacylglycerol)phosphate (BMP) is uniquely located to multivesicular bodies (MVBs) and late endosomes/lysosomes (LE/Lys), where it controls intra-lumenal vesicles (ILVs) formation. Upon fusion of MVBs with the membrane, ILVs will become exosomes. BMP thus regulates the correct addressing of cargoes to recycling, lysosomal degradation or extracellular exosomal export. Pathways regulated by BMP are profoundly altered in neurodegenerative diseases and levels of BMP are modified in these disorders -e.g., in human autopsy brain samples of late onset Alzheimer’s disease (LOAD). The ability to modulate BMP levels would open new perspectives in our understanding of the pathogenesis of these disorders and could provide new therapeutic options. The key obstacle to this approach is that the enzymes regulating BMP biosynthesis were so far unknown. Our lab recently identified an enzyme with phospholipase A2 (PLA2) activity -hereafter PLA2- regulating the limiting step of BMP synthesis in liposomes and in HeLa cell lines. Here we propose that modulating PLA2 levels and accompanying BMP levels could be an attractive strategy to modify pathological outcomes in LOAD. However, there is little that is known about PLA2 function and localization in the brain. In Aim 1, we will test the existence of a direct link between PLA2 protein levels and LOAD pathology in human autopsy brain samples. Specifically, we will use frozen samples from entorhinal cortex, hippocampus, prefrontal cortex and cerebellum of patients with severe or mild LOAD and compare them with unaffected control individuals. Using a combination of western blot and lipidomics, we will test (i) if protein levels of PLA2 are modified in pathology-prone brain regions of LOAD patients compared to controls, (ii) if protein levels of PLA2 correlate with disease progression and (iii) if BMP levels correlate with PLA2 levels. In Aim 2, we will gather evidence pertaining to the most disease-relevant brain cell type and biological outcomes to target in a future disease-modifying strategy. We will first identify the brain cell type localization of the PLA2 protein, using co-staining for PLA2 and different cell type markers in control human autopsy brain samples sections, in wild- type mouse brain sections and in microglia/astrocyte/neuron primary tri-cultures. In parallel, we will test whether modifying PLA2 protein levels leads to altered endolysosomal function and affects LOAD-relevant outcomes such as amyloid processing and exosomal release. We will down-regulate or overexpress Pla2 in mouse microglia/astrocyte/neuron primary tri-cultures and test whether it affects BMP levels, amyloid production and exosomal release, using a combination of confocal microscopy, western blot and Meso Scale Discovery (MSD) assays. If successful, the proposed studies will enable us to establish a strong baseline of experimental evidence on which we can build a robust disease-modifying strategy, to be investigated in a future larger application.

概括在调节细胞内转运途径的脂质中，双（单酰基甘油）磷酸酯（BMP）是独一无二的位于多泡体 (MVB) 和晚期内体/溶酶体 (LE/Lys)，控制腔内囊泡（ILV）形成后，MVB 与膜融合，ILV 将成为外泌体。调节货物对回收、溶酶体降解或细胞外外泌体输出的正确处理。在神经退行性疾病中，BMP 调节的通路发生了深刻的改变，BMP 水平也发生了变化。在这些疾病中进行了修改，例如，在晚发性阿尔茨海默病（LOAD）的人体尸检大脑样本中。调节 BMP 水平的能力将为我们理解 BMP 的发病机制开辟新的视角。这些疾病并可能提供新的治疗选择，这种方法的主要障碍是酶。我们的实验室最近发现了一种与磷脂酶 A2 相关的酶。 (PLA2) 活性（以下简称 PLA2）调节脂质体和 HeLa 细胞系中 BMP 合成的限制步骤。在这里，我们建议调节 PLA2 水平和伴随的 BMP 水平可能是一种有吸引力的方法。然而，人们对 PLA2 的功能知之甚少。在目标 1 中，我们将测试 PLA2 蛋白水平与大脑中的定位之间是否存在直接联系。具体来说，我们将使用来自内嗅的冷冻样本。重度或轻度 LOAD 患者的皮质、海马、前额叶皮质和小脑并进行比较与未受影响的对照个体结合使用蛋白质印迹和脂质组学，我们将测试 (i) 是否存在蛋白质。与对照组相比，LOAD 患者的病理倾向脑区的 PLA2 水平发生了改变，(ii) 如果蛋白质 PLA2 水平与疾病进展相关，以及 (iii) BMP 水平是否与 PLA2 水平相关在目标 2 中，我们。将收集与疾病最相关的脑细胞类型和生物学结果有关的证据未来的疾病缓解策略我们将首先使用 PLA2 蛋白的脑细胞类型定位。在野生环境中对对照人类尸检脑样本切片中的 PLA2 和不同细胞类型标记物进行共染色类型小鼠脑切片和小胶质细胞/星形胶质细胞/神经元原代三培养物中，我们将同时测试是否。改变 PLA2 蛋白水平会导致内溶酶体功能改变并影响 LOAD 相关结果例如淀粉样蛋白加工和外泌体释放，我们将下调或过度表达小鼠中的 Pla2。小胶质细胞/星形胶质细胞/神经元原代三培养并测试其是否影响 BMP 水平、淀粉样蛋白产生和结合使用共聚焦显微镜、蛋白质印迹和 Meso Scale Discovery (MSD) 进行外泌体释放如果成功，拟议的研究将使我们能够建立强有力的实验基线。我们可以根据这些证据制定强有力的疾病缓解策略，并在未来更大规模的研究中进行研究应用。