Rapid and Gentle Immunopurification of Brain Synaptic Vesicles

Abstract

Current methods to isolate synaptic vesicles (SVs), the organellar quanta of synaptic transmission, require highly specialized materials and up to 24 hours. These technical obstacles have thus far limited the study of SVs in models of synaptic function and pathophysiology. Here, we describe techniques for the rapid isolation of SVs by immunoprecipitation with widely available antibodies conjugated to magnetic beads. We report that the inexpensive rho1D4 monoclonal antibody binds SVs and show that elution with the 1D4 peptide yields native vesicles that are ≥ 10-fold purer than those obtained with classical techniques. These methods substantially widen the accessibility of SVs, enabling their purification in 60-90 minutes for downstream analyses including mass spectrometry and cryoelectron microscopy. Immunopurified SV preparations from mouse brain contained apolipoprotein E (ApoE), the low-density lipoprotein (LDL) receptor Lrp1, and enzymes involved in lipid metabolism, suggesting that SVs may play direct roles in lipid homeostasis and lipoprotein trafficking at the nerve terminal.Significance Statement Synaptic vesicles (SVs) are small organelles that form and recycle at nerve terminals to enable synaptic transmission. Much remains unknown about the processes that enable the formation and function of SVs. Moreover, nerve terminals appear to be particularly vulnerable to pathophysiologic processes underlying neurodegenerative diseases and schizophrenia. While techniques to purify synaptic vesicles thus have the potential to yield significant insights into physiology and pathophysiology of nerve terminals, current methods rely on either esoteric materials or expression of transgenes. This paper addresses these problems by establishing robust, efficient methods for SV purification using widely available materials, and it highlights several promising areas of future study arising from proteomic analyses of immunopurified SVs.

Highly fucosylated N-glycans at the synaptic vesicle and neuronal plasma membrane

“…Alongside synaptosomes, a highly pure population of SVs was isolated by immunoprecipitation (IP) with magnetic beads conjugated in-house to a monoclonal antibody against SV2 (Buckley and Kelly, 1985), according to recently described procedures ( Fig. 1A ) (Bradberry et al, 2022). These preparations were subjected to proteomic analysis by trypsin digestion and nano-flow liquid chromatography-tandem mass spectrometry (nLC-MS/MS) using an Orbitrap Eclipse mass spectrometer (Bradberry et al, 2022) ( Fig.…”

“…1E ). This represents the largest number of proteins detected in a highly pure SV preparation to date (Bradberry et al, 2022; Taoufiq et al, 2020). Label-free quantitation (LFQ) intensity scores of synaptosome and SV proteins were positively correlated ( Fig.…”

Synaptic vesicle proteins and ATG9A self-organize in distinct vesicle phases within synapsin condensates

“…However, consistent with our present findings, even in worms ATG9A did not appear to precisely colocalize with SVs: 1) its localization clearly diverged from the localization of SVs after perturbation of endocytosis 22,24 and 2) the localization of SV proteins and of ATG9A was differentially affected by genetic perturbation of the active/periactive zone protein Clarinet 24 . Moreover, while mass spectrometry analysis of purified SV fraction from rat brain detected ATG9A in such vesicles [25][26][27] , the amount of ATG9A detected was extremely low 26 . It was estimated that 4 copies of ATG9A were present in 100 vesicles 26 .…”

“…ATG9A is present in axon terminals where, together with other autophagy factors, it plays an important role in synapse development and homeostasis [22][23][24] . Proteomic studies have detected ATG9A (at low copy number) in purified SV fractions [25][26][27] . Moreover, recent imaging studies have shown that synaptically-localized ATG9A undergoes exo-endocytosis in response to activity in parallel with bona fide SV proteins 22,24 .…”

Synaptotagmin 7 outperforms synaptotagmin 1 to promote the formation of large, stable fusion pores via robust membrane penetration

“…Syt1 binds Ca 2+ with lower affinity (4), and responds to changes in [Ca 2+ ] with faster kinetics than syt7 (5). Syt1 is localized to SVs (2,(6)(7)(8), where it clamps spontaneous release (9,10) under resting conditions. Then, upon depolarization and Ca 2+ entry (11,12), syt1 functions to trigger and synchronize evoked release (9,(13)(14)(15).…”