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

Sammanfattning: 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.