Action potential-coupled Rho GTPase signaling drives presynaptic plasticity

Shataakshi Dube O'Neil,Erik J Soderblom,Scott H Soderling,Ryohei Yasuda,Bence Rácz,Yudong Gao,Walter Evan Brown

doi:10.7554/elife.63756

Abstract

In contrast to their postsynaptic counterparts, the contributions of activity-dependent cytoskeletal signaling to presynaptic plasticity remain controversial and poorly understood. To identify and evaluate these signaling pathways, we conducted a proteomic analysis of the presynaptic cytomatrix using in vivo biotin identification (iBioID). The resultant proteome was heavily enriched for actin cytoskeleton regulators, including Rac1, a Rho GTPase that activates the Arp2/3 complex to nucleate branched actin filaments. Strikingly, we find Rac1 and Arp2/3 are closely associated with synaptic vesicle membranes in adult mice. Using three independent approaches to alter presynaptic Rac1 activity (genetic knockout, spatially restricted inhibition, and temporal optogenetic manipulation), we discover that this pathway negatively regulates synaptic vesicle replenishment at both excitatory and inhibitory synapses, bidirectionally sculpting short-term synaptic depression. Finally, we use two-photon fluorescence lifetime imaging to show that presynaptic Rac1 activation is coupled to action potentials by voltage-gated calcium influx. Thus, this study uncovers a previously unrecognized mechanism of actin-regulated short-term presynaptic plasticity that is conserved across excitatory and inhibitory terminals. It also provides a new proteomic framework for better understanding presynaptic physiology, along with a blueprint of experimental strategies to isolate the presynaptic effects of ubiquitously expressed proteins.

Highlights

Dynamic tuning of neurotransmitter release in response to patterns of activity is a fundamental process that governs how experience modulates neural networks
We turned to a proximity-based proteomics approach, in vivo Biotin Identification, in which the promiscuous biotin ligase BioID2 is fused to a protein in a compartment of interest, and nearby biotinylated proteins are identified by mass spectrometry (Kim et al, 2016; Spence et al, 2019; Uezu et al, 2016)
BioID2-Synapsin was enriched in presynaptic boutons to Bassoon, an active zone marker, while the localization of BioID2 was indistinguishable from GFP, confirming it acts as a soluble fill (Figure 1—figure supplement 1D)

Summary

Introduction

Dynamic tuning of neurotransmitter release in response to patterns of activity is a fundamental process that governs how experience modulates neural networks. The actin cytoskeleton has long been implicated in many stages of the synaptic vesicle cycle that could modulate short-term plasticity, including exocytosis, endocytosis, vesicle trafficking, and reserve pool clustering (Cingolani and Goda, 2008; Rust and Maritzen, 2015) These potential roles have been controversial, as actin depolymerizing agents have enhanced, reduced, or had no effect on each of these processes depending on the study (Cole et al, 2000; Darcy et al, 2006; Gaffield et al, 2006; Gramlich and Klyachko, 2017; Lee et al, 2012; Morales et al, 2000; Sakaba and Neher, 2003; Sankaranarayanan et al, 2003). This challenges the prevailing view that the Rac1-Arp2/3 pathway functions largely at excitatory postsynapses, prompting re-evaluation of its mechanism in neurodevelopmental disorders

Results

Discussion

Materials and methods

Funding Funder National Science Foundation