14-3-3θ phosphorylation exacerbates alpha-synuclein aggregation and toxicity.

Aggregation of alpha-synuclein (αsyn) plays an integral role in Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). 14-3-3θ is a highly expressed brain protein with chaperone-like activity that regulates αsyn folding. 14-3-3θ overexpression reduces αsyn aggregation, transmission between cells, and neuronal loss, while 14-3-3 inhibition promotes αsyn pathology. We previously observed increased 14-3-3θ phosphorylation at serine 232 in human PD and DLB brains. Here we examine 14-3-3θ phosphorylation's effects on αsyn aggregation and toxicity. Using a paracrine αsyn model, we found that the non-phosphorylatable S232A 14-3-3θ protected while the phosphomimetic S232D 14-3-3θ failed to protect against αsyn paracrine toxicity. The S232A mutant reduced oligomerization of released αsyn while the S232D mutant did not. The S232D mutant showed significant reduction in αsyn binding compared to wildtype or S232A 14-3-3θ. Using knock-in mouse models expressing the S232A or S232D mutation in the cortex and hippocampus, we examined the impact of S232 phosphorylation on αsyn aggregation in the αsyn preformed fibril (PFF) model. Primary neurons from S232D mice showed increased αsyn inclusion formation compared to neurons from Cre control mice upon PFF treatment. In contrast, neurons from S232A mice showed reduced αsyn inclusions. αSyn PFF injection into the dorsolateral striatum induced higher αsyn inclusion numbers in the sensorimotor cortex of S232D mice compared to Cre control mice. In conclusion, 14-3-3θ phosphorylation at S232 interrupts the ability of 14-3-3θ to bind and regulate αsyn aggregation. Increased 14-3-3θ phosphorylation observed in human PD and DLB likely accelerates neurodegeneration in these disorders.