SUMMARY/ABSTRACT Parkinson’s disease (PD), dementia with Lewy bodies (DLB), multiple-system atrophy (MSA) and certain forms of Alzheimer’s disease (AD) are ‘synucleinopathies’ - brain diseases characterized by lesions (Lewy bod- ies/Lewy neurites) rich in α-synuclein (αS). Disease-modifying treatments are not available, in part due to a lack of insight into how native αS dynamics becomes aberrant. Our long-term goal is to understand αS biology in detail and to develop strategies to preserve/reestablish the normal physiological state and function of αS. αS in Lewy bodies/neurites is often phosphorylated on serine-129 (pS129), and the kinase(s) involved have been discussed as potential drug targets. However, pS129 may also have normal physiological role at synapses sup- ported by our observation that pS129 is reversibly induced by neural activity. Our overall objectives in this appli- cation are to (i) identify the synaptic mechanisms by which activity regulates pS129, and (ii) determine the rele- vance of pS129 for αS function at the synapse. Our central hypothesis is that polo-like kinase 2 (Plk2) phosphor- ylates αS at serine-129 in response to synaptic activity, thereby fine-tuning αS function. The rationale for this project is that understanding normal synaptic αS phosphorylation is likely to offer new insight for the development of strategies to preserve αS homeostasis, correct αS imbalance and quantify signatures of αS pathology. We propose the following specific aims: 1) Identify the mechanism(s) that govern reversible αS pS129 during neu- ronal activity. 2) Identify the role of reversible pS129 in fine-tuning αS function. Under the first aim, primary rodent cortical neuron cultures will be used to confirm Plk2 as the kinase that mediates pS129 during neuronal activity and to identify molecular changes in αS and Plk2 that may trigger pS129. Key findings will be confirmed in mouse hippocampal slices and in a mouse model of enriched environment. In the second aim, we will study the effects of pS129 on αS biology functionally in vitro and in vivo. Most importantly, we will characterize S129 phospho- deficient (S129A) and -mimicking (S129D) knock-in mouse models functionally with a special focus on dopamine release, using established methods in the Sulzer lab. The proposed research is innovative, because it focuses on dynamic αS S129 phosphorylation at αS’s normal locale (the synapse of mature neurons), considers synaptic activity as an important parameter, identifies key proteins, addresses functional consequences, and extends cell culture findings to in vivo. Whereas pS129 has been widely studied, most previous work focused on its role in pathological deposits. The contribution will be significant because it is expected to provide novel, paradigm- shifting insight into normal αS biology at the synapse. Corroborating that an αS modification commonly associ- ated with disease occurs normally, and understanding how and why, is an important step...