Endoplasmic Reticulum Dynamic Structural Changes in Neurons: The Fission-Fusion Phenomena

Abstract: The endoplasmic reticulum (ER) is crucial for protein synthesis and protein maturation, is involved in cell stress and serves in neurons as the major intracellular Ca2+ store. Neuronal ER forms a continuous network of cisterns and tubules extending from soma to a subset of dendritic spines. The continuity of ER structure is important for maintaining ER basic functions and necessary for proteins and ions to diffuse and equilibrate within its lumen. We show that ER in neurons can undergo rapid fission (=fragmentation) and subsequently fusion. This phenomenon was previously unknown in neurons. Our findings show that ER fission is induced during N-methyl D-aspartate (NMDA) receptor-mediated Ca2+ entry to the cell in murine primary cultures and hippocampal slice cultures. Using different pharmacological approaches, we demonstrate, that ER fission is triggered independently on Ca2+ from ER stores. Subsequently, we show that mild hypothermia, reported to be protective in experimental stroke models, enhances ER fragmentation. Finally, we validate the occurrence of rapid neuronal ER fission in an animal cardiac arrest model of cerebral ischemia. We assessed ER structure using confocal microscopy live cell and tissue imaging, 2-photon laser scanning microscopy and transmission electron microscopy (TEM). The fluorescence imaging was performed on murine primary cultures cotransfected to express cytosolic and ER-specific markers; hippocampal slices from transgenic mice expressing ER-specific marker; as well as in transgenic living animals. To characterize the fission-fusion in a quantitative way, we developed a new data analysis method based on fluorescence recovery after photobleaching (FRAP). Our data show that neuronal ER is a dynamic organelle. We propose a model of ER continuity, where ER is in equilibrium with fission-fusion events. Stimulation of NMDA receptors shifts the equilibrium towards the fragmentation, while inhibiting NMDA receptors promotes the continuous state of ER. We conclude that ER fission-fusion may be of importance in physiology and disease. The molecular machinery regulating the reversible changes in ER morphology remains unknown.