PROJECT SUMMARY The evolutionary expansion of the brain along the primate lineage involved unequal scaling of brain regions, with telencephalon size increasing dramatically more than that of midbrain and hindbrain. Coupled with increased human lifespan, this reconfiguration of brain structures may put undue burden on neurons whose target regions have expanded disproportionately. These vulnerable “joints” include the small populations of midbrain dopaminergic (DA) neurons that project to vast target fields in the cortex and striatum and contribute to human-enriched disorders such as Parkinson’s disease. In turn, human DA neurons may have evolved compensatory neuroprotective mechanisms while adapting to supplying an enlarged telencephalon, but few studies have examined the evolution of selective vulnerability or compensatory mechanisms in the human lineage. We have designed an interdisciplinary approach to study human-specific properties of DA neurons using interspecies stem cell-derived organoids, primary tissue from human, chimpanzee, and rhesus macaque, machine learning approaches to genomics data, and functional analysis of variants by CRISPR. Our approach will enable direct measurement of dynamic gene regulatory responses and candidate protective pathways to age-related oxidative stress that cannot be measured from post-mortem tissue alone. Convolutional neural nets will help decode a dynamic regulatory grammar of oxidative stress responses, enabling predictions of the effects of all human- specific variants. Interspecies tetraploid cell fusions further enable experimental analysis of the effects of these variants in their native genomic context, with CRISPRi supporting further validation. Finally, comparative loss of function screening in dopaminergic neurons exposed to stress pathways will reveal conserved and human-specific genetic dependencies in these vulnerable cell types. Through the successful completion of these studies, we will determine which genomic elements and genetic changes underlie oxidative stress-dependent responses in dopaminergic neurons laying the groundwork for further targeting these cellular protective mechanisms across cell types and age-related stressors. Ultimately, this approach is generalizable to other age- related stressors and vulnerable cell types.