Rapid recycling of glutamate transporters on the astroglial surface

  1. Piotr Michaluk  Is a corresponding author
  2. Janosch Peter Heller
  3. Dmitri A Rusakov  Is a corresponding author
  1. National Academy of Sciences, Poland
  2. Royal College of Surgeons Ireland - RCSI, Ireland
  3. University College London, United Kingdom

Abstract

Glutamate uptake by astroglial transporters confines excitatory transmission to the synaptic cleft. The efficiency of this mechanism depends on the transporter dynamics in the astrocyte membrane, which remains poorly understood. Here, we visualise the main glial glutamate transporter GLT1 by generating its pH-sensitive fluorescent analogue, GLT1-SEP. FRAP-based imaging shows that 70-75% of GLT1-SEP dwell on the surface of rat brain astroglia, recycling with a lifetime of ~22 s. Genetic deletion of the C-terminus accelerates GLT1-SEP membrane turnover while disrupting its surface pattern, as revealed by single-molecule localisation microscopy. Excitatory activity boosts surface mobility of GLT1-SEP, involving its C-terminus, metabotropic glutamate receptors, intracellular Ca2+ and calcineurin-phosphatase activity, but not the broad-range kinase activity. The results suggest that membrane turnover, rather than lateral diffusion, is the main 'redeployment' route for the immobile fraction (20-30%) of surface-expressed GLT1. This finding reveals an important mechanism helping to control extrasynaptic escape of glutamate.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for each corresponding Figure.

Article and author information

Author details

  1. Piotr Michaluk

    Nencki Institute of Experimental Biology, National Academy of Sciences, Warsaw, Poland
    For correspondence
    p.michaluk@nencki.edu.pl
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2306-3314
  2. Janosch Peter Heller

    FutureNeuro, Royal College of Surgeons Ireland - RCSI, Dublin, Ireland
    Competing interests
    The authors declare that no competing interests exist.
  3. Dmitri A Rusakov

    UCL Queen Square Institute of Neurology, University College London, London, United Kingdom
    For correspondence
    d.rusakov@ucl.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9539-9947

Funding

Wellcome Trust (212251_Z_18_Z)

  • Dmitri A Rusakov

H2020 European Research Council (857562)

  • Dmitri A Rusakov

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: Animal expeirments were carried out in accordance with the national guideline and the European Communities Council Directive 0f November 1986, the European Directive 2010/63/EU on the Protection of Animals used for Scientific Purposes, and the United Kingdom Home Office (Scientific Procedures) Act of 1986, under UK Home Office Project licence PPL707524.

Reviewing Editor

  1. Yukiko Goda, RIKEN, Japan

Publication history

  1. Received: November 11, 2020
  2. Accepted: April 15, 2021
  3. Accepted Manuscript published: April 16, 2021 (version 1)
  4. Version of Record published: April 27, 2021 (version 2)

Copyright

© 2021, Michaluk 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.

Metrics

  • 1,452
    Page views
  • 259
    Downloads
  • 10
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Piotr Michaluk
  2. Janosch Peter Heller
  3. Dmitri A Rusakov
(2021)
Rapid recycling of glutamate transporters on the astroglial surface
eLife 10:e64714.
https://doi.org/10.7554/eLife.64714

Further reading

    1. Neuroscience
    Kasra Manoocheri, Adam G Carter
    Research Article

    Connections from the basolateral amygdala (BLA) to medial prefrontal cortex (PFC) regulate memory and emotion and become disrupted in neuropsychiatric disorders. The diverse roles attributed to interactions between the BLA and PFC may reflect multiple circuits nested within a wider network. To examine these circuits, we first used retrograde and anterograde anatomy to show that the rostral BLA (rBLA) and caudal BLA (cBLA) differentially project to prelimbic (PL) and infralimbic (IL) subregions of the mouse PFC. Using ex vivo whole-cell recordings and optogenetics, we then assessed which neuronal subtypes are targeted, showing that rBLA preferentially drives layer 2 (L2) cortico-amygdalar (CA) neurons in PL, whereas cBLA drives layer 5 (L5) pyramidal tract (PT) neurons in IL. We next combined in vivo silicon probe recordings and optogenetics to confirm that cBLA mainly influences IL L5, whereas rBLA primarily activates PL L2, but also evokes polysynaptic activity in PL L5. Lastly, we used soma-tagged optogenetics to explore the local circuits linking superficial and deep layers of PL, showing how rBLA can engage L2 CA neurons to impact L5 PT neuron activity. Together, our findings delineate how subregions of the BLA target distinct networks within the PFC and differentially influence output from PL and IL.

    1. Cell Biology
    2. Neuroscience
    Arnau Llobet Rosell, Maria Paglione ... Lukas Jakob Neukomm
    Research Article

    Axon degeneration contributes to the disruption of neuronal circuit function in diseased and injured nervous systems. Severed axons degenerate following the activation of an evolutionarily conserved signaling pathway, which culminates in the activation of SARM1 in mammals to execute the pathological depletion of the metabolite NAD+. SARM1 NADase activity is activated by the NAD+ precursor nicotinamide mononucleotide (NMN). In mammals, keeping NMN levels low potently preserves axons after injury. However, it remains unclear whether NMN is also a key mediator of axon degeneration and dSarm activation in flies. Here, we demonstrate that lowering NMN levels in Drosophila through the expression of a newly generated prokaryotic NMN-Deamidase (NMN-D) preserves severed axons for months and keeps them circuit-integrated for weeks. NMN-D alters the NAD+ metabolic flux by lowering NMN, while NAD+ remains unchanged in vivo. Increased NMN synthesis, by the expression of mouse nicotinamide phosphoribosyltransferase (mNAMPT), leads to faster axon degeneration after injury. We also show that NMN-induced activation of dSarm mediates axon degeneration in vivo. Finally, NMN-D delays neurodegeneration caused by loss of the sole NMN-consuming and NAD+-synthesizing enzyme dNmnat. Our results reveal a critical role for NMN in neurodegeneration in the fly, which extends beyond axonal injury. The potent neuroprotection by reducing NMN levels is similar to the interference with other essential mediators of axon degeneration in Drosophila.