Genetically Encoded and Modular SubCellular Organelle Probes (GEM-SCOPe) reveal lysosomal and mitochondrial dysfunction driven byPRKNknockout

Abstract

Cellular processes including lysosomal and mitochondrial dysfunction are implicated in the development and progression of many diseases and disorders, including neurodegenerative diseases and cancer. However, there is limited ability to continuously monitor these sub-cellular processes in real-time, and standardized assays to quantify these differences are lacking. Quantitative visualization of mitochondrial and lysosomal networks is crucial to better understand how these organelles are dysregulated during disease pathogenesis. To address this gap, we developed GEM-SCOPe (Genetically Encoded and Modular SubCellular Organelle Probes), an expandable toolbox of genetically encoded fluorescent markers, introduced into cell cultures by lentiviral transduction, which can be used to interrogate changes in proliferation and lysosomal and mitochondrial function in cell models of disease pathogenesis and progression. Genetically encoded fluorescent markers are a powerful tool to consistently visualize subcellular components in live cells without disrupting cellular processes. The fluorophores in GEM-SCOPe are designed to specifically localize to the desired organelle and illuminate information on localization, distribution, organelle turnover, and intracellular oxidative stress. GEM-SCOPe is modular; it can be expanded to include existing or new fluorophores and targeted to other subcellular structures or cell types, increasing the potential for genetically encoded fluorescent markers to probe any cell-model system or subcellular network in live cell cultures. To validate and illustrate the translational functionality of GEM-SCOPe, we applied it to track and quantify sub-cellular pathophysiology changes associated with Parkinson's Disease mediated by loss of function knockout of the PRKN gene. We expressed GEM-SCOPe in a PRKN knockout induced pluripotent stem cell line, differentiated into astrocytes and neurons, enabling us to track and quantify in real-time disease-associated changes in cellular proliferation, lysosomal distribution, mitochondrial transport and turnover, and reactive oxygen species. Collectively, we demonstrate that GEM-SCOPe is a powerful panel of live-cell fluorescent probes that provide critical insight into the subcellular mechanisms underlying Parkinson's disease in human astrocytes and neurons. GEM-SCOPe can be applied to a diverse range of cellular models of disease to glean an understanding of the underlying mechanisms that promote disease onset and progression.