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

神经退行性疾病模型中鞘脂代谢在耦合溶酶体和线粒体功能障碍中的作用。

• 批准号: 2549729
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2549729
• 关键词: role; sphingolipid; metabolism; coupled; lysosome

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

研究背景线粒体和溶酶体是细胞代谢的重要调节因子，这两种细胞器的功能障碍与包括帕金森病（Parkinson 'sdisease，PD）在内的神经退行性疾病有关。最近的研究显示膜接触位点（MCS）在为蛋白质相互作用、信号传导事件、脂质交换和胆固醇稳态提供平台方面的复杂网络和重要性（Martello等人，2020年）。尼曼匹克C型（NPC）病（Vanier，2010）是一种进行性神经退行性疾病，其特征在于细胞水平上的晚期内体/溶酶体（LE/Lys）中的胆固醇积累，其已用于破译MCS的作用并研究固醇运输（Colaco等人，2020; Höglinger等人，2019年; Meng等人，2020;托雷斯等人，2017年）。NPC 1突变细胞与受损的线粒体分裂和溶酶体和线粒体中增加的胆固醇相关（Saffari et al.，2017;托雷斯等人，2017; Wos等人，2016年）。该小组先前的工作显示了在NPC 1缺陷细胞中LE/Lys：线粒体MCS的显著STARD 3依赖性扩增（Höglinger等人，2019年）。这种扩张可能与线粒体胆固醇从溶酶体输入触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系留蛋白ORPP 1 L的LE/Lys：ER MCS扩增逆转了NPC细胞模型中的胆固醇积累（Höglinger et al.，2019).葡萄糖脑苷脂酶（GBA 1）基因的突变被认为是PD的最重要的已鉴定遗传脆弱性因素，其中发展PD的风险增加20-30倍（Sidransky et al.，2009年）。GBA 1编码β-葡糖脑苷脂酶（GCase），其催化葡糖神经酰胺水解为葡萄糖和神经酰胺。GBA 1突变携带者PD患者表现出更严重的认知症状和更早的发病年龄（Do等人，2019年）。最近的研究表明，GBA 1突变PD患者来源的神经元具有延长的LE/LyS：线粒体MCS，这是由解束缚蛋白TBC 1D 15的调节缺陷引起的，该蛋白介导Rab 7 GTP水解以实现接触解束缚（Kim等人，2021年）。这种失调与患者来源的神经元中GBA 1溶酶体酶活性降低有关，并且可以通过用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，并且可以通过TBC 1D 15在PD患者衍生的GBA 1连接的神经元中进一步挽救。此外，我们小组的初步研究还显示，鞘氨醇激酶CRISPR KO细胞中LE/Lys：线粒体MCS增加，其中葡萄糖神经酰胺前体神经酰胺升高。有趣的是，除了转运胆固醇之外，最近显示NPC 1介导溶酶体鞘氨醇的流出（Altuzar et al.，2021），可能解释了NPC 1缺陷细胞中鞘脂的积累，并可能有助于扩大LE/Lys：线粒体MCS。我们这个博士项目的目的是表征与健康对照相比，来自NPC 1患者的iPSC神经元细胞中LE/Lys：线粒体MCS动力学和线粒体自噬。我们进一步的目的是研究鞘脂代谢和LE/Lys：线粒体MCS动力学之间的关系。目的目的1：LE/Lys的表征：NPC 1神经元细胞模型中的线粒体定位、动力学和MCS。目的2：鉴定影响溶酶体-线粒体MCS的化合物。目的3：使用体内斑马鱼模型验证结果