Development of Knockin Mouse Models for Study of a Novel Alpha-Synuclein Phosphorylation Site

PROJECT SUMMARY Synucleinopathies are associated with increased phosphorylation of specific α-synuclein residues, such as Serine 129 (pS129) in Parkinson's Disease. Recent work has suggested that phosphorylation of α-synuclein is also relevant to its physiological function in synaptic vesicle (SV) trafficking. For example, pS129 was shown to be neuronal activity-dependent and allows α-synuclein to differentially associate with lipid membranes to ultimately regulate synaptic transmission. To better understand the mechanisms of synucleinopathies, the impact of other α-synuclein post-translational modifications (PTMs) on both pathology and physiology must be determined. We are interested in investigating the impact of phosphorylation at Threonine 81 (pT81), a modification that was discovered, by unbiased proteomics, to be enriched in the midbrain of Multiple Systems Atrophy (MSA) patients. To investigate pT81, we developed and validated a phospho-specific polyclonal antibody against pT81 α-synuclein and confirmed the presence of pT81 α-synuclein in intranuclear, neuronal inclusions in MSA patient tissue. Interestingly, a recent phosphoproteomics screen revealed pT81 to be a novel, activity-dependent phosphorylation site on α-synuclein that modulates SV trafficking. Hence, we hypothesize that pT81 PTM, similarly to pS129, plays both a role in synucleinopathy pathology and physiological SV dynamics. Our long-term goal is to study pT81 in vivo to understand its physiological relevance as well as how it may contribute to pathology in MSA. To achieve this, we will use CRISPR/Cas9 editing to create a phosphorylation-null T81A α-synuclein knock-in (KI) mouse and a phosphorylation-mimetic T81E α-synuclein KI mouse (Aim 1). We will then begin to characterize these models by assessing heterozygous and homozygous KI mice for α-synuclein expression and phosphorylation at T81 and S129 (Aim 2). Currently there are limited in vivo tools to study the causal role of α-synuclein PTMs, which are widespread across synucleinopathies and may be crucial to synaptic function. By utilizing both a phospho-null and a phospho-mimetic mouse model, we can elucidate both the importance of this PTM to synaptic transmission as well as the effects of upregulated pT81 in MSA.