Parkinson Disease (PD) is a debilitating neurodegenerative disease, leading to progressive motor and cognitive dysfunction that affects 1% of the population over the age of 60. Although often considered a late onset disease, several lines of evidence suggest that disease onset occurs decades before motor symptoms arise. Prodromal PD symptoms, like intestinal dysmotility, anosmia, sleep disturbances, and depression may present decades prior to the onset of motor deficits, at which point a patient has lost ≥60% of their dopaminergic (DA) neurons from the substantia nigra (SN). Therefore, this proposal addresses a pivotal question in the study of late onset neurological disease. The overarching aim of this proposal is to define the timeline along which PD- related neurodegeneration begins, versus when it begins to produce quantifiable and potentially irreversible neurological effects. We will begin addressing this question by determining whether there is a critical time interval in which a PD causative mutation mediates its pathological effect. Indeed, PD mouse models that constitutively express disease-causing mutations often yield milder phenotypes than those induced later in life. The observation of this effect in multiple independent strategies points to the potential for developmental compensation rather than a technical or experimental phenomenon. Whether expression of PD-causing mutations mediate their effects in a time dependent manner, remains largely untested. By characterizing this window of pathological effect, our proposed work has the potential to inform a new wave of research involving therapeutic strategies and early disease screening. We propose to employ a tetracycline (Tet)-inducible model of the human alpha synuclein (SNCA) hA53T PD mutation, under the control of a DA neuron regulatory sequence (PITX3-IRES2-tTA/tetO-A53T) to study this question directly. To ensure the biological robustness of addressing this question with this Tet-Off model, we will first evaluate the latency between administration of the tetracycline analog, doxycycline (Dox), and repression of SNCA A53T transcript and protein levels, as well as between Dox removal/washout and SNCA A53T de- repression. Next, to determine whether the pathological effect of mutant SNCA is mediated throughout life or within a defined time window (e.g., gestation, postnatal, juvenile, or mid-adult life), we will activate expression of the mutant protein across five time windows: between mid-gestation (embryonic day 15.5) and postnatal day 21 (P21), between E15.5 and postnatal day 60 (P60), between P21 and mid-late adulthood (8-16 mos), between P60 and mid-late adulthood (8-16 mos), and mid-late adulthood (8-16 mos). These will be compared to controls expressing the mutant protein from E15.5 onwards and those in which the mutant protein remains inactivated. We will use well-established assays to evaluate the impact on behavioral, motor, and histological phenotypes. Our proposal will...