Abstract
Recently, Parkinson’s disease-associated α-synuclein (αS) has emerged as an important regulator for SNARE-dependent vesicle fusion. However, it is controversial if excessive accumulation of αS, even in the absence of aggregation, impairs neurotransmission. Here we use a single vesicle fusion assay with ms time resolution capable of dissecting the impact of αS on each step of membrane fusion. Unlike the previous results from various in vitro, cellular, and in vivo studies, we find that non-aggregated αS promotes vesicle merger even at exorbitant concentrations. The enhancement has been seen as much as 13 fold. Delving into the kinetics of the intermediate states for vesicle fusion reveals that αS stimulates vesicle docking without altering the dynamics of bilayer merger (lipid mixing). However, minute amounts of soluble aggregated species abolish SNARE-dependent bilayer merger completely. Thus, the results show that excessive accumulation of non-aggregated αS may not be toxic for neurotransmitter release.
Highlights
Neurotransmitter release is the foundation of thought in the central nervous system
DNA sequences encoding syntaxin-1A, VAMP2, SNAP-25, and αS were inserted into a pGEX-KG vector as N-terminal glutathione S-transferase (GST) fusion proteins
To investigate the effect of αS on SNARE-dependent vesicle fusion, we analyzed the merger of single v-SNARE VAMP2reconstituted vesicles (v-vesicles) to the supported bilayer reconstituted with t-SNAREs syntaxin-1A and SNAP25 (t-bilayer) with total internal reflection fluorescence (TIRF) microscopy (Figure 1A)
Summary
Neurotransmitter release is the foundation of thought in the central nervous system. The brain is comprised of networks of neurons joined together via synapses. Communication between these neurons is mediated by vesicle fusion releasing neurotransmitters at the synapses. Defects in the regulation of neurotransmission are believed to be the primary cause for many neurological diseases. The main cause for Schizophrenia is thought to be over-release of dopamine, while for Epilepsy it may be the under-release of adenosine and glycine (Boison et al, 2012). The key to a better understanding of neurological diseases may lie in a more complete understanding of the molecular machine responsible for synaptic vesicle fusion
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