PHASE SEPARATION OF SYNAPSIN-1: IMPLICATIONS FOR NEUROPSYCHIATRIC DISORDERS

Neuropsychiatric disorders (NPDs), including autism spectrum disorder (ASD) and epilepsy, are highly prevalent, yet remain poorly treated due to limited understanding of their underlying molecular mechanisms. Impaired synaptic transmission is a hallmark across NPDs, and recent studies have identified synapsin-1 (SYN1) variants in patients with NPDs, particularly ASD and epilepsy. SYN1 is the most abundant presynaptic phosphoprotein that undergoes liquid-liquid phase separation, forming biomolecular condensates that organize synaptic vesicles (SVs) at the synapse. Here, we investigate how reversible phosphorylation of SYN1 regulates SV clustering and how NPDs-associated variants disrupt the SV cycle. We combine coarse-grained molecular dynamics simulations with the Martini 3 force field for disordered proteins, in vitro experiments with purified proteins and model membranes, as well as functional validation in primary hippocampal neurons. Initial simulations indicate that phosphorylation of SYN1 at PKA and CaMKII sites leads to its conformational collapse at the membrane interface, consistent with results from reconstituted experimental systems. Functional analyses in synapsin total knock-out neurons using pHluorin-based assays further demonstrate that phosphorylation-dependent membrane binding is required for presynaptic SV accumulation and normal exocytosis. Examination of five SYN1 variants with high predicted pathogenicity (S79W, R420Q, A550T, T567A, Q555X) using biophysical assays and simulations revealed mutation-specific alterations in SYN1 density distributions and membrane interaction energies. Overall, our findings provide mechanistic insight into how SYN1 mutations perturb liquid-liquid phase separation and SYN1-SVs interactions, linking disrupted presynaptic organization to network hyperexcitability in NPDs.