Robo2 regulates synaptic oxytocin content by affecting actin dynamics
- Weizmann Institute of Science, Israel
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 showed 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.
Data availability
All source data files and codes were uploaded. Source data as summary tables is provided for all graphs and plots shown (Fig. 1E; 2B-E and G-J, 3E-H and J,K,M,N, 4D-F, 5O,R, 6I, 7C,D). R codes used for data import and analysis is provided.
Article and author information
Author details
Funding
Israel Science Foundation (1511/16)
- Savani Anbalagan
- Janna Blechman
- Michael Gliksberg
- Ludmila Gordon
- Gil Levkowitz
Israel Science Foundation (2137/16)
- Savani Anbalagan
- Janna Blechman
- Michael Gliksberg
- Ludmila Gordon
- Gil Levkowitz
Minerva Foundation ((Minerva Stiftung))
- Savani Anbalagan
- Janna Blechman
- Michael Gliksberg
- Ludmila Gordon
- Gil Levkowitz
United States-Israel Binational Science Foundation (2017325)
- Michael Gliksberg
- Gil Levkowitz
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Reinhard Jahn, Max Planck Institute for Biophysical Chemistry, Germany
Ethics
Animal experimentation: Experiments involving zebrafish were approved by the Weizmann Institute'sInstitutional Animal Care and Use Committee (protocol #27220516)
Version history
- Received: January 30, 2019
- Accepted: June 8, 2019
- Accepted Manuscript published: June 10, 2019 (version 1)
- Version of Record published: June 24, 2019 (version 2)
- Version of Record updated: October 5, 2020 (version 3)
Copyright
© 2019, Anbalagan et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Robo2 regulates synaptic oxytocin content by affecting actin dynamics
eLife 8:e45650.
https://doi.org/10.7554/eLife.45650
Further reading
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The gain-of-function mutation in the TALK-1 K+ channel (p.L114P) is associated with maturity-onset diabetes of the young (MODY). TALK-1 is a key regulator of β-cell electrical activity and glucose-stimulated insulin secretion. The KCNK16 gene encoding TALK-1 is the most abundant and β-cell-restricted K+ channel transcript. To investigate the impact of KCNK16 L114P on glucose homeostasis and confirm its association with MODY, a mouse model containing the Kcnk16 L114P mutation was generated. Heterozygous and homozygous Kcnk16 L114P mice exhibit increased neonatal lethality in the C57BL/6J and the CD-1 (ICR) genetic background, respectively. Lethality is likely a result of severe hyperglycemia observed in the homozygous Kcnk16 L114P neonates due to lack of glucose-stimulated insulin secretion and can be reduced with insulin treatment. Kcnk16 L114P increased whole-cell β-cell K+ currents resulting in blunted glucose-stimulated Ca2+ entry and loss of glucose-induced Ca2+ oscillations. Thus, adult Kcnk16 L114P mice have reduced glucose-stimulated insulin secretion and plasma insulin levels, which significantly impairs glucose homeostasis. Taken together, this study shows that the MODY-associated Kcnk16 L114P mutation disrupts glucose homeostasis in adult mice resembling a MODY phenotype and causes neonatal lethality by inhibiting islet insulin secretion during development. These data suggest that TALK-1 is an islet-restricted target for the treatment for diabetes.
