PROJECT SUMARY Parkinson’s Disease (PD) is a progressive neurodegenerative disease with 50,000-60,000 diagnoses annually and over 1 million Americans afflicted in total. PD-associated motor symptoms arise from the selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Because SNpc neurons send long- projecting axons to the striatum, this stereotypical neurodegeneration robs the striatum of crucial dopaminergic inputs and thereby renders an important motor feedback pathway ineffective. Alpha synuclein protein is known as the pathological hallmark of PD pathology. The function of alpha synuclein in the healthy brain is not completely understood, however it is transported down axons in abundance, is highly enriched in presynaptic terminals, and is believed to be responsible for the transmission and progression of PD pathology across different regions of the nervous system. Although researchers have learned a great deal about PD pathophysiology through cellular and animal models, the findings have had limited translational impact due to challenges in recapitulating the most disease-relevant attributes of human brain structure and function. In particular, there are limitations of current in vitro and in vivo models to recapitulate essential features of human disease related to axon pathophysiology and synuclein transmission. For instance, a key feature related to early SNpc vulnerability is that each dopaminergic neuron features a long-projecting axon with complex arborization that can total 15 feet in length within the striatum, incurring unique transport and metabolic needs of these neurons. This feature of long axonal projections from human derived dopaminergic neurons – the human source ensuring a genetic endowment capable of developing and responding to synucleinopathy – projecting to a striatal neuronal source has been absent in preclinical models of PD thereby underrepresenting the role of axonopathy and metabolic susceptibility in PD pathogenesis. To address this need, we have developed the first tissue engineered nigrostriatal pathway (TE-NSP) recapitulating key elements of the native pathway: discrete human stem cell derived, phenotypically-controlled neuronal populations connected by long- projecting axonal tracts. This project will validate TE-NSPs as the first PD model featuring anatomically inspired microtissue, and then apply this novel platform for the study of PD axonopathy, mechanisms of synuclein transmission, and pharmacological interventions to block axon-mediated spread of pathological alpha-synuclein across discrete brain structures. We will first demonstrate that TE-NSPs appropriately recapitulate the relevant systems-level architecture by identifying all source and target cell types, characterizing synaptic formation with presynaptic and postsynaptic markers, and demonstrating input-output based on evoked dopamine release (AIM 1). We will then model PD via the addition of exogenous alpha synuclein...