Mechanisms of Neuronal Loss Mediated by mTORC1-TFEB Deregulation in Human iPSC Model of GBA1-Associated Parkinsons Disease

GBA1 相关帕金森病人类 iPSC 模型中 mTORC1-TFEB 失调介导的神经元丢失机制

• 批准号: 10282462
• 负责人: Ola A. Awad
• 金额: $ 42.49万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10282462
• 关键词: Apoptosis; Apoptotic; Autophagocytosis; Biochemical; Biogenesis; Biological Assay; Cell Death; Cell Line; Cells; Cellular Stress; Data; Development; Endoplasmic Reticulum; Enzymes; Equilibrium; Excision; Experimental Models; FRAP1 gene; Fluorescence; Functional disorder; Future; Gaucher Disease; Genes; Genetic; Glucosylceramides; Goals; Homeostasis; Human; Hyperactivity; Induced pluripotent stem cell derived neurons; Inherited; Link; Lipids; Measures; Mediating; Mitochondria; Modeling; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nuclear Translocation; Parkinson Disease; Pathogenesis; Pathologic; Pathway interactions; Patients; Pharmacology; Phosphorylation; Play; Proteins; Risk Factors; Signal Transduction; Sphingolipids; Structure; Testing; age related; alpha synuclein; base; biological adaptation to stress; dopaminergic neuron; effective therapy; endoplasmic reticulum stress; genetic risk factor; induced pluripotent stem cell; mTOR inhibition; mutant; neuron loss; novel; prevent; response; stem cell model; stem cells; transcription factor

PROJECT SUMMARY: Mutations in the GBA1 gene are the single, most frequent genetic risk factor for Parkinson's disease (PD), however the mechanisms how these mutations contribute to PD development are not fully understood. Using patients' neurons derived from induced-pluripotent stem cells (iPSCs), we recently uncovered that biallelic GBA1-mutations result in hyperactivity of the mammalian target of rapamycin complex1 (mTORC1), which negatively regulates the transcription factor EB (TFEB). TFEB is not only the master regulator of the autophagy-lysosomal pathway but also a critical regulator of cell fate. TFEB maintains mitochondrial structural and functional integrity and prevents endoplasmic reticulum (ER) stress. Both the mitochondria and ER play key roles in sensing and reacting to cellular stress and disruption of their functions leads to activation of apoptotic signals and neuronal death. While GBA1 mutations are known to cause mitochondrial and ER dysfunction in various experimental models, the underlying mechanisms remain unclear. We hypothesis that in GBA1-associated PD, TFEB deregulation by mTORC1 results in mitochondrial and ER dysfunction, which leads to neuronal death. The overall goal of this proposal is to determine the effects mTORC1-TFEB deregulation on mitochondrial homeostasis and ER stress, and to identify mechanisms of the consequent neuronal loss in GBA1-associated PD. We generated dopaminergic (DA) neurons form PD patients' iPSC lines harboring heterozygous GBA1 mutations and the corresponding isogeneic, gene- edited lines. Using this model, we will determine the involvement of mTORC1-TFEB deregulation on mitochondrial and ER alterations in PD. We will use biochemical and fluorescence-based assays to measure mitochondrial biogenesis, mitophagy, ER stress levels and apoptosis in PD DA neurons in both basal conditions and following pharmacological and genetic modulation of mTORC1-TFEB activity. We expect to detect decreased mitophagy, accumulation of defective mitochondrial and increased ER stress levels, which mediates apoptosis in PD DA neurons. We also anticipate that suppressing mTORC1 and restoring TFEB activity can reverse these alterations. Our results will define the relationship between GBA1 mutations, deregulation of mTORC1-TFEB axis, and neuronal loss, thus providing mechanistic understanding of neurodegeneration in PD. Moreover, Our use of PD iPSCs lines with GBA1 mutations and the corresponding gene-edited ones enables us to obtain mechanistic findings and link the results to the inherited mutations. Our novel model and approach make the results relevant to PD patients and facilitate future development of effective therapies capable or preventing neuronal loss. In the long term, this project will pave the way to develop TFEB-based therapies capable of preventing neuronal loss, which will have significant impact not only on PD but on many other neurodegenerative disorders as well.

项目总结： GBA1基因突变是帕金森病的单一、最常见的遗传风险因素 然而，这些突变如何促进帕金森病发生的机制尚不完全清楚。 利用患者来自诱导多能干细胞(IPSCs)的神经元，我们最近发现 双等位基因GBA1突变导致哺乳动物雷帕霉素复合体靶标1(MTORC1)的过度活性， 负调控转录因子EB(TFEB)。TFEB不仅是世界银行的主要监管者 自噬-溶酶体途径也是细胞命运的关键调节因子。TFEB维持线粒体结构 和功能的完整性，并防止内质网(ER)应激。线粒体和内质网都发挥作用 在感知和反应细胞应激和功能中断中的关键作用导致激活 细胞凋亡信号和神经元死亡。虽然已知GBA1突变会导致线粒体和内质网 在各种实验模型中，其潜在机制尚不清楚。我们假设在 MTORC1对GBA1相关的PD、TFEB的去调节导致线粒体和内质网功能障碍，从而 会导致神经元死亡。这项提案的总体目标是确定mTORC1-TFEB的影响 线粒体动态平衡和内质网应激的解除调控及其机制 GBA1相关帕金森病的神经元丢失。我们从帕金森病患者的IPSC系产生了多巴胺(DA)能神经元 含有杂合的GBA1突变和相应的等基因、基因编辑的品系。使用这个 模型，我们将确定mTORC1-TFEB失控对线粒体和内质网改变的影响 在警局。我们将使用生化和基于荧光的分析来测量线粒体的生物发生， 基础状态和后续状态下PD-DA神经元的有丝分裂、内质网应激水平和细胞凋亡 MTORC1-TFEB活性的药理和遗传调节。我们希望能检测到吞丝分裂的减少， 线粒体缺陷的积累和内质网应激水平的增加，介导了PD DA的细胞凋亡 神经元。我们还预计，抑制mTORC1和恢复TFEB活性可以逆转这些 改装。我们的结果将定义GBA1突变、mTORC1-TFEB放松调控之间的关系 轴和神经元的丢失，从而提供了对帕金森病神经变性的机制理解。而且，我们的 使用带有GBA1突变的PD IPSCs系和相应的基因编辑的系使我们能够获得 机械的发现，并将结果与遗传突变联系起来。我们的新模式和新方法使 与帕金森病患者相关的结果，并有助于未来开发能够或预防的有效治疗方法 神经元丢失。从长远来看，该项目将为开发基于TFEB的疗法铺平道路， 防止神经元丢失，这不仅对帕金森病有重大影响，而且对许多其他疾病也有重大影响 神经退行性疾病也是。

项目成果

Deregulation of mTORC1-TFEB axis in human iPSC model of GBA1-associated Parkinson's disease.

• DOI: 10.3389/fnins.2023.1152503
• 发表时间: 2023
• 期刊: FRONTIERS IN NEUROSCIENCE
• 影响因子: 4.3
• 作者: Mubariz, Fahad;Saadin, Afsoon;Lingenfelter, Nicholas;Sarkar, Chinmoy;Banerjee, Aditi;Lipinski, Marta M.;Awad, Ola
• 通讯作者: Awad, Ola