PROJECT SUMMARY Parkinson’s disease (PD) is projected to double in prevalence by 2040 and yet there are no current therapeutic approaches that delay or stop disease progression. A broad role for leucine-rich repeat kinase 2 (LRRK2) mutations in familial and idiopathic PD has emerged, elevating its status to a central disease target. Ultimately, prevention of LRRK2 neurotoxicity will require a detailed understanding of the key mechanisms driving neurodegeneration. One of the most frequent neuronal defects associated with the common LRRK2 G2019S mutation in vitro is a loss of neurite length and complexity. Yet, the nature of these defects in vivo, their underlying cause, and their relationship to dopamine neuron death are all unknown. This creates a major roadblock to understanding disease etiology. An unbiased screen for genetic modifiers of LRRK2 G2019S neurodegeneration lead to the discovery of three genes; prospero, cut and pbl, which all have roles in neurite outgrowth and maintenance. This generates the hypothesis that prospero, cut and pbl drive LRRK2 G2019S- induced dopaminergic neurite defects, and that these defects are necessary and sufficient for dopamine neuron death in aged animals. In the proposed studies, the impact of LRRK2 G2019S on dopaminergic neurite growth and maintenance across aging in Drosophila will be determined using conditional transgenics that overexpress LRRK2 G2019S. LRRK2 G2019S expression will be induced either throughout the life span or restricted to development or aging to determine how this impacts dopaminergic neurite defects observed in aged flies. Mechanisms involving prospero, cut and pbl in LRRK2 G2019S neurite defects through altered cytoskeletal regulation and their role in neuronal death will be examined. The contribution of the mammalian orthologs of prospero (PROX1), cut (CUX1) and pbl (ECT2) to substantia nigra dopamine neuron loss will be assessed in a rat adenoviral model of LRRK2 G2019S-induced neurodegeneration. Successful completion of the proposed research will contribute to the understanding of (i) molecular mechanisms of LRRK2 G2019S- induced neurite defects in vivo (ii) the nature of these defects across development and aging in vivo (iii) whether these defects are necessary and sufficient for age-related dopamine neuron death and (iv) whether the mammalian orthologs of the identified modifiers also contribute to LRRK2 G2019S neurodegeneration. This contribution is expected to be significant because it will provide a major advance in understanding the mechanisms driving LRRK2 G2019S neurodegeneration in PD. This proposal incorporates a number of conceptual and technological innovations to achieve a detailed study of the nature and mechanisms of LRRK2- related neurite defects, their dynamics across aging and their connection to established PD-related phenotypes.