ABSTRACT Parkinson's disease (PD) is one of the most common neurodegenerative disorders. It is characterized by the progressive loss of dopamine neurons in the substantia nigra (SN) pars compacta, and their projections onto striatal neurons and the accumulation of α-Synuclein aggregates often into Lewy bodies. The pathogenesis of PD is not fully elucidated but there is vast evidence supporting complex loops of neuroinflammatory cascades, mitochondrial dysfunction, degenerating neurons, sustained microglial activation and other pathophysiological mechanisms that together amplify a relentless progression towards neuronal loss in the nigra and beyond. α- Synuclein (α-S) aggregates are implicated either directly in serving as a template that is transmitted transneuronally and seeding further aggregation, and/or in amplifying the neuroinflammatory loop, leading in both cases to neuronal dysfunction and death. To date, there are no therapeutic options that lead to the regeneration of lost neurons or to the restoration of circuitry. Our group has pioneered the derivation of functional dopamine neurons from human embryonic stem cells (hES) and we have just completed a Phase 1 clinical trial for the bilateral intrastriatal grafting of these cells. There is much excitement about the restorative potential of stem cell derived neurons in PD, but there remain multiple challenges. Here we propose to study the impact of microenvironmental alterations in the brain in the context of 2 different mouse models: the 3K mouse model which expresses a triple mutant form of α-synuclein based on the human E46K mutation, and exhibits histological hallmarks of PD as well as progressive motor and other behavioral abnormalities; the second model consists of the intrastriatal injection of preformed α-synuclein fibrils (PFF) which spread transneuronally through the brain to form pathogenic α-synuclein inclusions, leading to loss of DA neurons and behavioral deterioration. These models are predicated on two different hypotheses and will serve as great tools to study inflammation and its impact on behavior. In addition, we will graft the mice with the same hES cell derived dopamine neurons used in the clinical trial to analyze the impact of the microenvironment on the neurons' survival and phenotype, as well as on their ability to rescue behavior. We will capture grafted cells as well as host microglia and astrocytes at key timepoints during the in vivo lifespan of the grafts to establish dynamic maps of cell lineages, maturation, microglial and astrocytic phenotypes and potentially activation of neurotoxic signals. In the last aim, we will engineer the human ES cells to delete the SNCA gene encoding α-synuclein in an attempt at increasing the resistance of the grafts to neurotoxicity and potentially the transmission of pathogenic α- synuclein. Data obtained in this proposal will serve to further enhance our understanding of neuro-inflammation in different PD microenvironments an...