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

项目概要： GBA 1基因突变是帕金森病最常见的遗传风险因素 (PD)然而，这些突变如何促进PD发展的机制尚未完全了解。 使用源自诱导多能干细胞（iPSC）的患者神经元，我们最近发现 双等位基因GBA 1突变导致雷帕霉素复合物1的哺乳动物靶标（mTORC 1）的过度活性， 其负调节转录因子EB（TFEB）。TFEB不仅是 自噬-溶酶体途径，而且也是细胞命运的关键调节剂。TFEB维持线粒体结构 和功能完整性，并防止内质网（ER）应激。线粒体和内质网 在感知和反应细胞应激和破坏其功能的关键作用，导致激活 凋亡信号和神经元死亡。虽然已知GBA 1突变会导致线粒体和ER 在各种实验模型中，功能障碍的潜在机制仍不清楚。我们假设， GBA 1相关的PD，由mTORC 1引起的TFEB失调导致线粒体和ER功能障碍， 导致神经元死亡本提案的总体目标是确定mTORC 1-TFEB的影响 线粒体稳态和内质网应激的失调，并确定随之而来的机制， GBA 1相关PD中的神经元丢失。我们从PD患者的iPSC细胞系中产生多巴胺能（DA）神经元 携带杂合GBA 1突变的品系和相应的等基因的基因编辑品系。使用此 模型，我们将确定mTORC 1-TFEB失调对线粒体和ER改变的参与 在警局我们将使用生物化学和基于荧光的分析来测量线粒体的生物合成， 在基础条件和以下条件下，PD DA神经元的线粒体自噬、ER应激水平和凋亡 mTORC 1-TFEB活性的药理学和遗传学调节。我们希望检测到线粒体自噬减少， 有缺陷的线粒体的积累和ER应激水平的增加，其介导PD DA中的细胞凋亡 神经元我们还预计，抑制mTORC 1和恢复TFEB活性可以逆转这些变化。 改变。我们的研究结果将确定GBA 1突变、mTORC 1-TFEB失调和GBA 1基因突变之间的关系。 轴，和神经元的损失，从而提供机制的理解，在PD神经变性。而且我们 使用具有GBA 1突变的PD iPSC系和相应的基因编辑的PD iPSC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。我们的新模式和方法使 与PD患者相关的结果，并促进未来开发能够或预防 神经元缺失从长远来看，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PD而且对许多其他疾病都有重大影响。 神经退行性疾病

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.