Aggregation of α-synuclein scaffolds RNA G-quadruplexes assembly leading to progressive neurodegeneration

Synucleinopathies are triggered by the aggregation of α-synuclein (αSyn), leading to progressive neurodegeneration. However, the intracellular mechanism of αSyn aggregation remains unclear. Here we show that assembly of RNA G-quadruplexes (rG4s) form scaffolds for αSyn aggregation, contributing to neurodegeneration. Purified αSyn binds rG4s directly through the N-terminus. rG4 itself undergoes phase separation and assembly by Ca2+, accelerating the sol-gel phase transition of αSyn. In αSyn preformed fibrils (PFF)-treated neurons, rG4s assembly composed of synaptic mRNAs co-aggregates with αSyn upon Ca2+ excess influx into cytoplasm, eliciting synaptic dysfunction. Artificial assembly of rG4s using an optogenetic approach evokes αSyn aggregation and neuronal dysfunction. Administration of 5-aminolevulinic acid (5-ALA), a prodrug of protoporphyrin IX (PpIX) that prevents phase separation of rG4s, attenuating αSyn aggregation, neurodegeneration, and progressive motor deficits in PFF-injected synucleinopathy mice. Together, assembly of rG4s due to dysregulation of intracellular Ca2+ homeostasis accelerates αSyn phase transition and aggregation may contribute to pathogenesis of synucleinopathies. Primary mouse cortical neurons (mCTXN) treated with vehicle or α-Syn PFF were crosslinked on ice cold RNase free PBS by irradiation with UV light (254 nm) at 150 mJ/cm2. Cells were harvested, lysed, and precleared on DynabeadsTM Protein G (Invitrogen) for 1 hour at 4 ℃. The precleared lysates were incubated at 4°C for three hours in a protein G magnetic beads immobilized with normal rabbit IgG (for control) or BG4 with anti-DYKDDDDK tag antibody. Purified protein-bound RNA fraction was subjected to library preparation using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB). All libraries were sequenced on a NextSeq 500 sequencer (illumina).