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

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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.