About one million Americans live with Parkinson's Disease (PD) which is characterized by progressive loss of subpopulations of nigral midbrain dopaminergic neurons (DNs), leading to motor impairment and other debilitating conditions. Familial PD genes show broad expression in the brain and neurodegeneration in PD can be widespread; however, it is unclear why nigral DNs show such exquisite vulnerability compared to other cell types, including other DN populations. Post-mortem studies suggest that oxidative stress (OS) contributes to familial and sporadic PD. Reactive oxygen species (ROS) are important signaling molecules but high levels of intracellular ROS will damage DNA, lipids and proteins. High energy needs and dopamine metabolism may explain increased ROS, OS and the unique vulnerability of nigral DNs but human-relevant model systems are required to rigorously test this hypothesis. There is an urgent need to develop experimental systems to better understand nigral DN vulnerability, identify novel disease-relevant signaling mechanisms, and improve molecular subtyping and patient stratification. Our long-term goal is to understand the vulnerability of nigral DNs through the interplay of genetics, cell type specific functions that confer vulnerability and quantifiable phenotypes to identify new therapeutic targets. In support of this goal, we have developed knock-in human pluripotent stem cell (hPSC) reporter lines to identify and isolate tyrosine hydroxylase (TH)-positive DNs from large-scale organoid spin cultures. Using CRISPR mutagenesis we created isogenic loss-of-function models of early-onset autosomal recessive PD (PARKIN-/-, DJ1-/- and ATP13A2-/-) in TH-reporter cell lines. We detected dysregulation of mitochondrial proteins, significantly increased OS and cell death in isogenic PD cell lines in midbrain DNs, but not in isogenic WT-control DNs. To understand nigral DN vulnerability we propose to use our isogenic reporter PD model and single-cell RNA sequencing approaches of human midbrain, hypothalamic and forebrain DNs to identify populations of cells that show increased vulnerability to OS and cell death and identify differentially affected DN populations. Expression and network analysis will identify cellular functions that confer vulnerability. We have developed genetic tools to distinguish primary dysregulation from emerging phenotypes to further the mechanistic understanding of genotype-phenotype interactions. Using innovative CRISPR-activation and inhibition technologies we will test identified candidate genes for their potential to ameliorate OS phenotypes and cell death in our PD model. Our model is conceptually and technically innovative and will illuminate common and unique pathways that confer vulnerability or protection in nigral DNs and propose novel strategies for prevention and treatment to improve the lives of patients and their caregivers.