NMDA Receptor Stimulation Induces Reversible Fission of the Neuronal Endoplasmic Reticulum

Krzysztof Kucharz,Håkan Toresson,Morten Krogh,Ai Na Ng,Fabien Tell

doi:10.1371/journal.pone.0005250

Krzysztof Kucharz, Håkan Toresson + Show 3 more

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https://doi.org/10.1371/journal.pone.0005250

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Journal: PloS one	Publication Date: Apr 21, 2009
Citations: 57	License type: CC BY 4.0

Affiliation: Lund University

Abstract

With few exceptions the endoplasmic reticulum (ER) is considered a continuous system of endomembranes within which proteins and ions can move. We have studied dynamic structural changes of the ER in hippocampal neurons in primary culture and organotypic slices. Fluorescence recovery after photobleaching (FRAP) was used to quantify and model ER structural dynamics. Ultrastructure was assessed by electron microscopy. In live cell imaging experiments we found that, under basal conditions, the ER of neuronal soma and dendrites was continuous. The smooth and uninterrupted appearance of the ER changed dramatically after glutamate stimulation. The ER fragmented into isolated vesicles in a rapid fission reaction that occurred prior to overt signs of neuronal damage. ER fission was found to be independent of ER calcium levels. Apart from glutamate, the calcium ionophore ionomycin was able to induce ER fission. The N-methyl, D-aspartate (NMDA) receptor antagonist MK-801 inhibited ER fission induced by glutamate as well as by ionomycin. Fission was not blocked by either ifenprodil or kinase inhibitors. Interestingly, sub-lethal NMDA receptor stimulation caused rapid ER fission followed by fusion. Hence, ER fission is not strictly associated with cellular damage or death. Our results thus demonstrate that neuronal ER structure is dynamically regulated with important consequences for protein mobility and ER luminal calcium tunneling.

Highlights

Activation of glutamate receptors triggers a multitude of intracellular signaling pathways important for many aspects of CNS physiology and disease
To explore endoplasmic reticulum (ER) fragmentation further, we transfected hippocampal cultures to express a fluorescent protein targeted to the ER (EGFP-ER or DsRed2-ER (RedER) where the fluorescent protein is flanked by an ER-targeting sequence and a KDEL ER retention signal previously shown to label neuronal ER [28,29,30]) and a cytosolic fluorescent protein of another color (EGFP or DsRed2)
Our experiments show that activation of the NMDA receptor is necessary and sufficient for inducing neuronal ER fission in dissociated and organotypic culture

Summary

Introduction

Activation of glutamate receptors triggers a multitude of intracellular signaling pathways important for many aspects of CNS physiology and disease. Some of these signaling events terminate on the endoplasmic reticulum (ER) and are important for many aspects of brain function [1,2,3,4,5,6,7,8]. The continuity of the ER is important for its normal function as a calcium store as well as for its role in the secretory pathway. ER continuity allows mature proteins to move to the specialized ER exit sites from which they are trafficked to the Golgi [15,16,17,18]

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