How Serine-129 Phosphorylation Status Affects the Spreading of α-Synuclein Pathology in Vivo: a Study in Knock-in Animals

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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 in detail how normal αS biology goes awry 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, our recent research also suggests a normal physiological role of pS129 in regulating synaptic activity (pS129 is reversibly induced by neural activity). Our overall objectives in this application are to (i) identify the effect of pS129 on the seeded intraneuronal aggregation of αS, and (ii) to determine the relevance of pS129 for the spreading of αS pathology in animal brains. Our central hypothesis is that both lack and excess of pS129 may trigger αS aggregation in the first place (because normal αS dynamics are perturbed in the absence of the “right” level of pS129). The subsequent effect on spreading is exploratory. The rationale for this project is that understanding both normal and seeded 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. Taking advantage of our novel S129 phospho-deficient (S129A) and - mimicking (S129D) knock-in (KI) mouse models, we propose the following specific aims: 1) Effects of PFFs and patient-derived seed on αS aggregation in cultured WT, S129AKI, and S129DKI neurons. 2) Effects of PFFs and patient-derived seed on αS aggregation and pathogenic spread in WT, S129AKI, and S129DKI mice. Under Aim 1, primary rodent cortical neuron cultures (WT, S129AKI, and S129DKI) will be used to determine the seeding effects of PFFs and patient-derived brain extracts± synaptic activity. Synaptotoxicity mechanisms will be studied in hippocampal slices of all genotypes. In Aim 2, we will determine the effects of pS129 on αS pathogenic spread in our animal models in vivo. As one outcome, we hope to have a comprehensive view on initiation vs. spreading of pathology as a function of pS129. The proposed research is innovative because it employs novel rodent KI models of pS129 loss and pS129 excess. To our knowledge, such models have never been treated with αS “seed” before. A systemic analysis of pS129 on initiation and spreading of abnormal αS biology has not been done. Our combination of cellular, brain slice, and animal models is also innovative. Our contribution will be significant because it is expected to provide novel, paradigm-shifting insight into how normal αS biology is per- turbed in disease, and how the perturbation spreads within the brain. Corroborating that pS129 plays a key role in disease initiation and spreading, and understanding how and why, is an important step towards a comprehen- sive view of αS in health and disease with major implications for drug and biomarker development.