Deciphering the metabolism of LBPA and its function in the endolysosomal system

描述(申请人提供)：内溶酶体系统缺陷越来越多地被视为神经退行性疾病的关键病理特征，如阿尔茨海默病(AD)和帕金森病。一种新的假说是，这些疾病中出现的慢性内溶酶体缺陷损害了溶酶体的降解能力，导致通常通过内吞或自噬途径靶向这些细胞器的各种溶酶体货物的异常积聚。这些货物不仅包括一系列易于聚集的蛋白质或多肽(例如，Abeta、异常的α-突触核蛋白和tau)，而且还包括脂类，如胆固醇和鞘磷脂。最近，我们的实验室采用了一种被称为“脂质组学”的系统方法，使用最先进的质谱学从健康和疾病组织中提取了数百种脂质。使用这项技术，我们在阿尔茨海默病患者的脆弱脑区发现了显著的脂质变化，在Niemann-Pick C型(NPC)小鼠模型中，这是一种侵袭性溶酶体储存障碍，与AD有一些共同的致病过程，以及在载脂蛋白衍生的胆固醇异常积累的其他情况下。这种脂质被称为溶二磷脂酸(LBPA)或双(单酰甘油)磷酸，这是一种非典型的磷脂，专门富含在多囊内体和溶酶体中，在那里它进一步集中在富含胆固醇的腔内小泡(ILV)上。LBPA以前被认为可以促进内溶酶体内ILV的形成，调节载脂蛋白衍生的胆固醇的储存和分布，并通过刺激水解酶促进溶酶体的降解。结合这些研究，我们的脂体学数据不仅确定LBPA是与内溶酶体功能障碍相关的疾病的候选生物标记物，而且它们还表明这种磷脂在这些细胞器的生理学和病理生理学以及在给药载脂蛋白衍生的胆固醇方面都发挥着关键作用。不幸的是，目前缺乏工具来了解这种内溶酶体脂的确切(病理)生理作用，并操纵其水平来评估其治疗潜力。这一障碍背后的主要原因是，调节这种难以捉摸的磷脂合成和降解的酶是未知的。因此，这项建议的主要目标是(I)利用全基因组RNAi筛选和脂质组学相结合的方法，识别LBPA代谢酶以及更广泛地说，对LBPA水平进行正向或负向调节的基因；以及(Ii)评估各种类型的LBPA操作对正常神经元以及NPC1突变小鼠神经元溶酶体功能的影响。我们预计，我们的研究将对了解LBPA在溶酶体内生理学中的作用至关重要，并可能对与溶酶体功能障碍相关的疾病产生重大影响。

DESCRIPTION (provided by applicant): Defects in the endolysosomal system are increasingly viewed as key pathological features of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease. An emerging hypothesis is that chronic endolysosomal defects occurring in these disorders compromise the degradative capacity of lysosomes, causing the aberrant accumulation of a variety of lysosomal cargoes that are targeted to these organelles generally through the endocytic or autophagy pathway. These cargoes not only include a range of aggregate-prone proteins or peptides (e.g., Abeta, aberrant alpha-synuclein and tau), but also lipids, such as cholesterol and sphingolipids. Recently, our lab has employed a systems-based approach called "lipidomics" to profile hundreds of lipids from healthy and diseased tissue using state-of-the-art mass spectrometry. With this technology, we identified a striking lipid alteration in vulnerable brain regions from patients with AD, in a mouse model of Niemann-Pick type C (NPC), an aggressive lysosomal storage disorder that shares some pathogenic processes in common with AD, as well as in other instances where apolipoprotein-derived cholesterol is aberrantly accumulating. This lipid is called lysobisphosphatidic acid (LBPA) or bis(monoacylglycero) phosphate, an atypical phospholipid that is specifically enriched in the multivesicular endosomes and lysosomes, where it is further concentrated on cholesterol-rich intralumenal vesicles (ILVs). LBPA has been previously suggested to promote the formation of ILVs in the endolysosomal compartment, to regulate the storage and distribution of apolipoprotein-derived cholesterol, and facilitate lysosomal degradation by stimulating hydrolases. Together with these studies, our lipidomic data not only identify LBPA as a candidate biomarker for disorders associated with endolysosomal dysfunction, but they also suggest a critical role for this phospholipid both in the physiology and the pathophysiology of these organelles as well as in the administration of apolipoprotein-derived cholesterol. Unfortunately, tools are currently lacking to understand the precise (patho)physiological roles of this endolysosomal lipid and manipulate its levels to assess its therapeutic potential. The primary reason behind this roadblock is that the enzymes mediating the synthesis and degradation of this elusive phospholipid are unknown. The main goals of this proposal are thus (i) to identify the LBPA-metabolizing enzymes and more generally, the genes positively or negatively regulating the levels of LBPA using a combination of genome-wide RNAi screen and lipidomics; and (ii) to assess the impact of various types of LBPA manipulations on lysosomal function in normal neurons as well as in neurons derived from Npc1 mutant mice. We anticipate that our studies will be critical to understand the role of LBPA in endolysosomal physiology with potentially major implications for disorders associated with lysosomal dysfunction.