Action potential-coupled Rho GTPase signaling drives presynaptic plasticity

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.

Presynaptic Rac1 controls synaptic strength through the regulation of synaptic vesicle priming

“…Subsequently, the RRP replenishment rates are described by the time constants of fits. Using the slope of the line fit, we found that Rac1 -/- calyces had faster SV replenishment rates and larger steady-state EPSC amplitudes at 50 Hz train stimulation which is similar to observations in primary hippocampal cultures at 20 and 40 Hz train stimulation (O’neil et al, 2021). In contrast, at 500 Hz train stimulation, there was no difference between Rac1 +/+ and Rac1 -/- calyces in SV replenishment rates or steady-state EPSC amplitudes.…”

“…Rac1 is found in the presynaptic terminal (Doussau et al, 2000; O’neil et al, 2021) and regulates synaptogenesis, axon guidance, and neuronal development (Xu and Chen, 2019; Zhang et al, 2021). In this study, we aimed to elucidate Rac1’s presynaptic function in controlling synaptic transmission and plasticity independent of its role in regulating synapse development and maturation at the calyx of Held/MNTB synapse.…”

“…We specifically abated Rac1 at a distinct developmental time point, two days after hearing onset where neuronal circuit properties are well defined and the calyx of Held is functionally mature (Englitz et al, 2009; Sonntag et al, 2009, 2011; Borst and Soria Van Hoeve, 2012). Although Rac1 was ablated at 10 days in vitro (O’neil et al, 2021), the corresponding developmental stage to in vivo hippocampal neurons is unclear. In addition, it is unknown to what extent the artificial in vitro circuit properties determine synaptic transmission and plasticity characteristics.…”

“…Rac1 is involved in multiple processes which control synaptogenesis, axon guidance, neuronal development, and postsynaptic function to regulate neuronal circuit function (Bai et al, 2015). Although Rac1 is expressed in both the pre and postsynaptic compartment (Threadgill et al, 1997; Doussau et al, 2000; Kumanogoh et al, 2001; O’neil et al, 2021), its presynaptic role in regulating synaptic transmission is not well understood. However, recent work using primary cultured hippocampal neurons proposed that presynaptic Rac1 is a negative regulator of SV replenishment (O’neil et al, 2021), though at which steps in the SV lifecycle it exerts its regulatory role is unknown.…”

“…Although Rac1 is expressed in both the pre and postsynaptic compartment (Threadgill et al, 1997; Doussau et al, 2000; Kumanogoh et al, 2001; O’neil et al, 2021), its presynaptic role in regulating synaptic transmission is not well understood. However, recent work using primary cultured hippocampal neurons proposed that presynaptic Rac1 is a negative regulator of SV replenishment (O’neil et al, 2021), though at which steps in the SV lifecycle it exerts its regulatory role is unknown. Finally, how Rac1 regulates the temporal dynamics of RRP release and replenishment in a native neuronal circuit remains elusive.…”