BMP-dependent pathways and Alzheimer's disease

摘要 在调节细胞内转运途径的脂质中，磷酸二(单甘油)(BMP)是唯一的 定位于多囊泡小体(MVB)和晚期内小体/溶酶小体(LE/Lys)，在那里它控制腔内 小泡(ILV)形成。当MVBS与膜融合后，ILV将成为外体。因此，BMP 规定货物的正确寻址，以回收、溶酶体降解或胞外胞外输出。 在神经退行性疾病中，BMP调节的通路发生了深刻的变化，BMP的水平 在这些疾病中进行修改--例如，在迟发性阿尔茨海默病(LOAD)的人类尸检大脑样本中。 调节BMP水平的能力将为我们理解骨质疏松症的发病机制打开新的视角 这些疾病，并可能提供新的治疗选择。这种方法的关键障碍是酶 到目前为止，对BMP生物合成的调控尚不清楚。我们实验室最近鉴定出一种磷脂酶A2 (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，我们认为调节PLA2水平和伴随的BMP水平可能是一个有吸引力的 修改负荷中的病理结果的策略。然而，人们对PLA2功能知之甚少 以及在大脑中的定位。在目标1中，我们将测试PLA2蛋白水平和 在人类尸检脑组织样本中装载病理学。具体地说，我们将使用内脏的冷冻样本 轻、重度负荷患者大脑皮质、海马体、前额叶、小脑的变化及比较 与未受影响的对照个体。利用蛋白质印迹和脂质组学的组合，我们将测试(I)蛋白质是否 与对照组相比，LOAD患者病理易感脑区的PLA2水平发生改变：(Ii)IF蛋白 PLA2水平与疾病进展相关；(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的证据作为目标 未来的疾病修正策略。我们将首先确定PLA2蛋白的脑细胞类型定位，使用 对照尸检脑组织切片中PLA2和不同细胞类型标记物的联合染色 小鼠脑切片和小胶质细胞/星形胶质细胞/神经元原代三代培养。同时，我们将测试是否 改变PLA2蛋白水平导致内溶酶体功能改变并影响负荷相关结局 如淀粉样蛋白加工和胞外体释放。我们将下调或过度表达小鼠的PLA2 小胶质细胞/星形胶质细胞/神经元原代三培养并检测其是否影响BMP水平，淀粉样蛋白的产生和 使用共聚焦显微镜、蛋白质印迹和中尺度发现(MSD)相结合的方法释放胞外体 化验。如果成功，拟议的研究将使我们能够建立一个强大的实验基线 我们可以建立强有力的疾病改善战略的证据，将在未来进行更大规模的调查 申请。

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.