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[Journal of Molecular Biology]The Role of α-Synuclein in SNARE-mediated Synaptic Vesicle Fusion, Volume 435, Issue 1, 15 January 2023, 167775
Gyeongji Yoo, Nam Ki Lee
Neuronal communication depends on exquisitely regulated membrane fusion between synaptic vesicles and presynaptic neurons, which results in neurotransmitter release in precisely timed patterns. Presynaptic dysfunctions are known to occur prior to the onset of neurodegenerative diseases, including Parkinson’s disease. Synaptic accumulation of α-synuclein (α-Syn) oligomers has been implicated in the pathway leading to such outcomes. α-Syn oligomers exert aberrant effects on presynaptic fusion machinery through their interactions with synaptic vesicles and proteins. Here, we summarize in vitro bulk and single-vesicle assays for investigating the functions of α-Syn monomers and oligomers in synaptic vesicle fusion and then discuss the current understanding of the roles of α-Syn monomers and oligomers in synaptic vesicle fusion. Finally, we suggest a new therapeutic avenue specifically targeting the mechanisms of α-Syn oligomer toxicity rather than the oligomer itself.
Figure 1. Processes of SNARE-mediated vesicle fusion A proposed SNARE-mediated fusion pathway at the synapse. Individual SNAREs are on separate membranes. Target membrane t-SNAREs (Syntaxin1 in blue and SNAP25 in red, depicted together), are attached to the plasma membrane, and vesicle (v-)SNAREs (VAMP2 in light blue) are on the synaptic vesicle. A) The synaptic vesicle and the plasma membrane are in the vicinity of each other. B) The synaptic vesicle is docked at the plasma membrane via SNARE complex assembly. C) After intermediate states including hemifusion, a fusion pore is formed. D) Fusion pore is expanded to a large pore, resulting in the complete merger of the vesicle to the presynaptic membrane and neurotransmitter release.
Figure 2. Schematics of in vitro bulk and single-vesicle fusion assays. A) Bulk vesicle fusion assay using fluorescently labeled lipids. When t-vesicles doped with the FRET donor dye DiI (T) and v-vesicles doped with the FRET acceptor dye DiD (V) fuse through SNARE complex formation, the lipids from the two types of vesicles are mixed. The degree of lipid mixing can be monitored by the increase of the FRET efficiency between DiI and DiD dyes. The FRET efficiency at each time point is calculated by the equation E = IDA/(IDD + IDA), where IDD and IDA denote the total donor fluorescence signal and the total acceptor fluorescence signal, respectively. B) Surface-immobilized single-vesicle fusion assay detects the docking and lipid mixing between two types of proteoliposomes. V-vesicles labeled with the acceptor dyes are attached on the surface via interactions between biotin-neutravidin. The fluorescence signals of v-vesicles are detected by the CCD camera. When v- and t-vesicle membranes approach each other, a small increase in FRET efficiencies occurs (less than 0.25). Upon lipid mixing, the acceptor dyes are mixed with the donor dyes in the t-vesicles, which increases the FRET efficiency to above 0.6. C) Diffusion-based single-vesicle fusion assay. (Left panel) The excitation laser is tightly focused to obtain a small confocal excitation volume (∼fL). While the fluorescently labeled proteoliposome passes through the excitation volume, it is excited alternately by the two lasers. (Middle panel) According to the source of the excitation laser and fluorescent probes, schematic descriptions of fluorescent bursts are shown. (Right panel) Two-dimensional E (FRET efficiency)–S (stoichiometry) graph obtained by ALEX. The E–S graph is used to obtain the subpopulations of unreacted T (green square), unreacted V (red square), docked vesicles (purple square), and fused vesicles (yellow square).
Figure 3. A proposed model of the functions of the α-Syn monomers and oligomers in SNARE-mediated vesicle fusion. A) Physiological condition: Native α-Syn binds to the synaptic vesicle membrane and vesicular SNARE (v-SNARE) and can assemble into non-toxic multimeric forms when bound to the membrane. The membrane-bound α-Syn has been found to promote SNARE complex assembly at the synapse and cluster synaptic vesicles to maintain a reserve pool in the vicinity of the active zone and regulate the neurotransmitter release. B) Pathological condition: Large α-Syn oligomers induce clustering of synaptic vesicles severely by binding to v-SNAREs, thereby blocking the fusion of synaptic vesicles with the plasma membrane. In addition, the association of α-Syn oligomers with the membrane has been reported to permeabilize and disrupt the membrane.