Robo2 regulates synaptic oxytocin content by affecting actin dynamics

Abstract

The regulation of neuropeptide level at the site of release is essential for proper neurophysiological functions. We focused on a prominent neuropeptide, oxytocin (OXT) in the zebrafish as an in vivo model to visualize and quantify OXT content at the resolution of a single synapse. We found that OXT-loaded synapses were enriched with polymerized actin. Perturbation of actin filaments by either cytochalasin-D or conditional Cofilin expression resulted in decreased synaptic OXT levels. Genetic loss of robo2 or slit3 displayed decreased synaptic OXT content and robo2 mutants displayed reduced mobility of the actin probe Lifeact-EGFP in OXT synapses. Using a novel transgenic reporter allowing real-time monitoring of OXT-loaded vesicles, we show that robo2 mutants display slower rate of vesicles accumulation. OXT-specific expression of dominant-negative Cdc42, which is a key regulator of actin dynamics and a downstream effector of Robo2, led to a dose-dependent increase in OXT content in WT, and a dampened effect in robo2 mutants. Our results link Slit3-Robo2-Cdc42, which controls local actin dynamics, with the maintenance of synaptic neuropeptide levels.

The Protein Arginine Methyltransferase PRMT8 and Substrate G3BP1 Control Rac1-PAK1 Signaling and Actin Cytoskeleton for Dendritic Spine Maturation

“…To examine actin dynamics, we performed fluorescence recovery after photobleaching (FRAP) in neurons expressing mCherry-LifeAct. FRAP of LifeAct in the dendritic spine is largely reduced by the F-actin stabilization drug jasplaki-nolide (Rocca et al, 2013), indicating its utility to monitor actin turnover in neurons as previously shown (Njoo et al, 2015;Gokhale et al, 2016;Tong et al, 2018;Anbalagan et al, 2019). Depletion of PRMT8 by either shRNA or gene knockout significantly reduced LifeAct recovery in dendritic spines, as indicated by the decrease in dynamic fraction (Figure 5D; Figure S4), which suggests slower F-actin turnover (George et al, 2015;Anbalagan et al, 2019).…”

Rho GTPases Signaling in Zebrafish Development and Disease

“…Indeed, mice lacking Cdc42 present defects in postsynaptic plasticity, while its loss of function in presynaptic sensory neurons affects synapse formation [81,82]. Zebrafish was also used as in vivo model to elegantly dissect the role of Cdc42 in actin dynamics at the resolution of a single synapse in a living vertebrate animal [83]. The study investigated the release of the neuropeptide oxytocin from large dense core vesicles in zebrafish and showed a crucial role for Cdc42, downstream of Robo2, in maintaining synaptic oxytocin levels by regulating synaptic actin dynamics.…”

“…The study investigated the release of the neuropeptide oxytocin from large dense core vesicles in zebrafish and showed a crucial role for Cdc42, downstream of Robo2, in maintaining synaptic oxytocin levels by regulating synaptic actin dynamics. Indeed, Robo2 acts as a GTPase activating protein (GAP) for Cdc42 since it promotes GTP hydrolysis and Cdc42 expression level correlated with oxytocin level in synapses [83].…”

Developmental effects of oxytocin neurons on social affiliation and processing of social information

“…Unlike the ablated OXT cells, which recovered over time, the deficits in DA neurons observed in early OXT-ablated larvae persisted through adulthood, as adult fish that had To demonstrate that at the time of their ablation, OXT neurons physically interact with the affected DA neurons, we examined whether OXT neurons form synapses on DA-ergic clusters of the PrT and TP (Figure 5). We employed a double transgenic line, Tg(oxt:Gal4;UAS:Sypb-EGFP), which genetically expresses the synaptic marker, synaptophysin-GFP in OXT neurons (Anbalagan et al, 2019), and visualized synaptic contacts onto TH positive DA-ergic neurons (Figure 5). To ensure that these are indeed bona-fide OXT releasing synapses, we also co-stained these fish with an anti-OXT antibody (Blechman et al, 2018;Anbalagan et al, 2019) (Figure 5C-D).…”

“…We employed a double transgenic line, Tg(oxt:Gal4;UAS:Sypb-EGFP), which genetically expresses the synaptic marker, synaptophysin-GFP in OXT neurons (Anbalagan et al, 2019), and visualized synaptic contacts onto TH positive DA-ergic neurons (Figure 5). To ensure that these are indeed bona-fide OXT releasing synapses, we also co-stained these fish with an anti-OXT antibody (Blechman et al, 2018;Anbalagan et al, 2019) (Figure 5C-D). We found OXT projections forming multiple synapses onto the DA neurons of the PrT and the TP (Figure 5C,D1,D2).…”