The role of sphingolipid metabolism in coupled lysosome and mitochondrial dysfunction in neurodegenerative disease models.

线粒体和溶酶体都是细胞代谢的关键调节因子，这两个细胞器的功能障碍与包括帕金森病（PD）在内的神经退行性疾病有关。最近的研究表明，膜接触位点（MCS）在为蛋白质相互作用、信号事件、脂质交换和胆固醇稳态提供平台方面具有复杂的网络和重要性（Martello et al., 2020）。Niemann Pick型c （NPC）病（Vanier, 2010）是一种进行性神经退行性疾病，在细胞水平上以晚期内体/溶酶体（LE/Lys）的胆固醇积累为特征，已被用于解释MCS的作用和研究胆固醇运输（Colaco等人，2020；Höglinger等人，2019；孟等人，2020；Torres等人，2017）。NPC1突变细胞与线粒体分裂受损以及溶酶体和线粒体胆固醇升高有关（Saffari等人，2017；Torres等人，2017；Wos等人，2016）。该小组之前的工作表明，在npc1缺陷细胞中，LE/Lys：线粒体MCS的stard3依赖性扩增惊人（Höglinger et al., 2019）。这种扩张可能与从溶酶体进口线粒体胆固醇引发线粒体功能障碍有关。该小组最近的工作还表明，通过表达LE/Lys系栓蛋白ORPP1L来扩增LE/Lys:ER MCS，可以逆转鼻鼻癌细胞模型中的胆固醇积累（Höglinger等人，2019）。葡萄糖脑苷酶（GBA1）基因突变被认为是帕金森病最重要的遗传易感性因素，患帕金森病的风险增加20-30倍（Sidransky et al., 2009）。GBA1编码β -葡萄糖脑苷酶（GCase），该酶催化糖基神经酰胺水解为葡萄糖和神经酰胺。GBA1突变携带者PD患者认知症状更严重，发病年龄更早（Do et al., 2019）。最近的研究表明，GBA1突变PD患者衍生的神经元具有延长的LE/Lys：线粒体MCS，这是由解系蛋白TBC1D15的缺陷调节引起的，该蛋白介导Rab7 GTP水解以实现接触解系（Kim et al., 2021）。这种失调与患者来源的神经元中GBA1溶酶体酶活性降低有关，可以通过GCase调节剂增加酶活性来挽救。这些缺陷导致线粒体分布和功能被破坏，可以通过TBC1D15进一步挽救PD患者来源的gba1连接神经元。此外，本课组前期研究还发现鞘氨酸激酶CRISPR KO细胞中LE/Lys：线粒体MCS升高，其中葡萄糖神经酰胺前体神经酰胺升高。有趣的是，除了运输胆固醇外，NPC1最近还被证明可以介导溶酶体鞘磷脂的分泌（Altuzar等，2021），这可能是NPC1缺陷细胞中鞘脂积累的原因，并可能导致LE/Lys：线粒体MCS的扩大。我们这个博士项目的目的是表征来自NPC1患者的iPSC神经元细胞的LE/Lys：线粒体MCS动力学和线粒体自噬，并与健康对照进行比较。我们将进一步研究鞘脂代谢与LE/Lys：线粒体MCS动力学之间的关系。目的1:NPC1神经元细胞模型中LE/Lys：线粒体定位、动力学和MCS的表征。目的2：鉴定影响溶酶体-线粒体MCS的化合物。目的3：利用斑马鱼体内模型验证结果

BackgroundBoth mitochondria and lysosomes are key regulators of cellular metabolism and dysfunction of these two organelles has been associated with neurodegenerative diseases including Parkinson's disease (PD). Recent studies show the complex network and importance of membrane contact sites (MCS) in providing platforms for protein interactions, signaling events, lipid exchange and cholesterol homeostasis (Martello et al., 2020). Niemann Pick type-C (NPC) disease (Vanier, 2010) is a progressive neurodegenerative disease characterized on a cellular level by cholesterol accumulation in the late endosomes/lysosomes (LE/Lys), that has been used to decipher the role of MCS and study sterol trafficking (Colaco et al., 2020; Höglinger et al., 2019; Meng et al., 2020; Torres et al., 2017). NPC1 mutant cells are associated with impaired mitochondrial fission and increased cholesterol in lysosomes and mitochondria (Saffari et al., 2017; Torres et al., 2017; Wos et al., 2016). Previous work by the group showed striking STARD3-dependent expansion of LE/Lys:mitochondria MCS in NPC1-deficient cells (Höglinger et al., 2019). This expansion may be linked to mitochondrial cholesterol import from lysosomes triggering mitochondrial dysfunction. Recent work from the group also showed that LE/Lys:ER MCS expansion by expression of LE/Lys tethering protein ORPP1L, reverses cholesterol accumulation in NPC cellular models (Höglinger et al., 2019).Mutations of the glucocerebrosidase (GBA1) gene is considered the most important identified genetic vulnerability factor for PD, where the risk of developing PD is increased 20-30 fold (Sidransky et al., 2009). GBA1 encodes Beta-glucocerebrosidase (GCase) that catalyses the hydrolysis of glucosylceramide to glucose and ceramide. GBA1 mutation carrier PD patients exhibit more severe cognitive symptoms and earlier age of onset (Do et al., 2019). Recent study showed that GBA1 mutant PD patient derived neurons have prolonged LE/Lys:mitochondria MCS caused by defective modulation of the untethering protein TBC1D15, which mediates Rab7 GTP hydrolysis for contact untethering (Kim et al., 2021). This dysregulation was linked to decreased GBA1 lysosomal enzyme activity in patient derived neurons and could be rescued by increasing enzyme activity with a GCase modulator. These defects resulted in disrupted mitochondrial distribution and function and could be further rescued by TBC1D15 in PD patient derived GBA1-linked neurons. In addition, preliminary studies by our group also showed increased LE/Lys:mitochondria MCS in sphingosine kinase CRISPR KO cells, where the glucosylceramide precursor ceramide is elevated. Interestingly, in addition to transporting cholesterol, NPC1 was recently shown to mediate the egress of lysosomal sphingosine (Altuzar et al., 2021), likely accounting for the accumulation of sphingolipids in NPC1-deficient cells and possibly contributing to the expanded LE/Lys:mitochondria MCS.Our aim for this PhD project is to characterize LE/Lys:mitochondria MCS dynamics and mitophagy in iPSC neuronal cells derived from NPC1 patients compared with healthy controls. We further aim to study the relationship between sphingolipid metabolism and LE/Lys:mitochondria MCS dynamics. AimsAim 1: Characterization of LE/Lys:mitochondria positioning, dynamics and MCS in NPC1 neuronal cell model. Aim 2: Identify compounds that affect lysosome-mitochondria MCS.Aim 3: Validation of results using in vivo zebrafish model