Effects of 14-3-3θ phosphorylation vesicular transport and alpha-synuclein aggregation - Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB) are progressive, incurable neurodegenerative disorders marked by pathological aggregation of alpha-synuclein (αsyn). Although extensive evidence indicates that αsyn aggregation causes neuronal dysfunction and loss, the detailed cellular mechanisms regulating αsyn toxicity are elusive. Understanding of homeostatic mechanisms that prevent αsyn aggregation in normal brains will inform the development of new disease-modifying therapies. One such promising target is 14-3-3θ. 14-3-3 proteins are highly expressed proteins that mediate neuronal function through protein-protein interactions (PPIs). The 14-3-3θ isoform is of particular importance in PD and DLB: 14-3-3θ overexpression reduces αsyn aggregation and toxicity, while 14-3-3θ inhibition exacerbates αsyn toxicity in αsyn models. We found an increase in phosphorylated 14-3-3θ at S232 in human PD and DLB, which correlated with cognitive decline. These data point to 14-3-3θ phosphorylation as a key contributor to αsyn pathology. Understanding the consequences of 14-3-3θ phosphorylation is critical to unlocking the potential of 14-3-3θ therapeutics. Molecular dynamics simulations reveal critical changes in the binding pocket upon S232 phosphorylation that are predicted to destabilize 14-3-3θ PPIs and thus disrupt its functions. Affinity-purified mass spectrometry from knock-in mice expressing the nonphosphorylatable S232A or the phosphomimetic S232D 14-3-3θ mutant showed reduced binding to several proteins critical to vesicular transport. In support of this, we found that retrograde axonal transport of acidic vesicles in S232D neurons is impaired. S232 phosphorylation also reduced αsyn binding and increased αsyn aggregation and toxicity in cellular αsyn models. Based on these data, we hypothesize that aberrant 14-3-3θ phosphorylation enhances vulnerability to αsyn by destabilizing critical PPIs. We predict 14-3-3θ phosphorylation 1) disrupts the 14-3-3θ/αsyn interaction needed to prevent αsyn aggregation, and 2) destabilizes 14-3-3θ/transport protein complexes needed to prevent αsyn accumulation. A promising therapeutic approach are stabilizers of 14-3-3 PPIs, several of which are currently in development as therapeutics for spinal cord injury. Fusicoccin-A (FC-A), a non-specific 14-3-3 PPI stabilizer, shows protection in αsyn models. In Aim 1, we will examine the structural interaction between 14-3- 3θ and αsyn, the impact of S232 phosphorylation on this PPI, and the impact of phosphorylation on αsyn aggregation and toxicity in vivo. In Aim 2, we will test the impact of S232 phosphorylation on axonal transport of AVs and lysosomes and targeting of αsyn into these vesicles. In both aims, we will use the non-specific PPI stabilizer FC-A as a tool compound to determine if augmentation of 14-3-3θ PPIs destabilized by 14-3-3θ phosphorylation can reduce αsyn pathology. Completion of the structural and functional studies in our proposal will enable discovery of clinically useful drugs to enhance 14-3-3θ PPIs for PD and DLB.
