Parkinson?s disease (PD) is a severe, second most common neurodegenerative disorder which is still poorly understood and has few current treatment options. The clinical phenotype of PD is caused by the selective degeneration of dopaminergic neurons in the substantia nigra pars compacta in the ventral midbrain. Early- onset PD accounts for 4-10% of all PD patients and autosomal recessive mutations in DJ1 have been associated with some of these PD cases. DJ1 is a mitochondrial protein encoded by the PARK7 gene, and plays a role in transcriptional regulation, kinase activity regulation, protein ubiquitination and oxidative stress. Mutations in DJ1 have been associated with mitochondrial dysfunction seen in PD, however the exact mechanism of how DJ1 mutations contribute to PD pathogenesis remains unclear. Recently, human induced pluripotent stem cell (hiPSC)-based PD models generated insight into the pathobiology of mutations in a number of other PD- associated genes, such as LRKK2, SNCA, Parkin and PINK1. However, no such human in vitro model exists for studying the impact of DJ1 mutations on mitochondrial function and PD etiology. Here we propose to combine hiPSC-technology with TALEN- and CRISPR/Cas9-based gene editing approaches to; 1) generate a cohort of isogenic DJ1 mutation containing lines with either the c.317_322del or c.T497C mutation; generate 2 different mitochondrial function iPSC reporter lines (GFP-LC3B and HyPer-GFP) to assay mitophagy/autophagy and mitochondrial morphology/ROS production. Upon dopaminergic (DA) differentiation of gene edited isogenic lines, samples will be collected at iPSC, midbrain floorplate progenitor and mature DA neuron stage, and used for transcriptional profiling. Assays analyzing ROS production, mitochondrial movement and morphology will be used to evaluate mitochondrial function in gene edited DJ1 mutation and control iPSC-derived DA neurons. DA neuron differentiation of mitochondrial reporter lines with gene edited DJ1 mutations will provide additional insights into the impact of DJ1 mutations on mitochondrial function in PD. The outcome of this work will not only provide new insights into DJ1 function in mitochondrial function, but also provide a powerful new resource for probing mitochondrial function in neurodegenerative disease.
DJ1-associated Parkinson?s disease (PD) is a severe, second most common neurodegenerative disorder which is still poorly understood and has few current treatment options. A major challenge in studying PD is to develop disease models that faithfully reproduce human disease and until recently it has been difficult to specifically model these human neurons in culture for molecular analyses. Here we propose to combine human induced pluripotent stem cell technology with state-of-the-art gene editing technology to generate a cohort of isogenic DJ1 patient, as well as mitochondrial function reporter iPSC lines, which may not only open up new opportunities for probing DJ1-PD disease mechanisms but also establish a general platform for modeling (CNS) disorders associated with mitochondrial dysfunction.
