Endoplasmic Reticulum Dynamic Structural Changes in Neurons: The Fission-Fusion Phenomena
(2010) In Lund University Faculty of Medicine Doctoral Dissertation Series 2010:129.- 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... (More) - 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. (Less) - Abstract (Swedish)
- Popular Abstract in Swedish
Det endoplasmatiska retiklet (ER) är nödvändigt för proteinsyntes och
posttranslationell proteinmodifiering. ER är också iblandat i cellstress och
utgör den största intracellulära kalciumkällan i nervceller. Neuronalt ER bildar
ett kontinuerligt nätverk som består av dels rörliknande förbindelser (tubules)
och större cisterner. Att nätverket utgör en kontinuerlig lumen är en viktig
egenskap som gör att proteiner och kalciumjoner kan röra sig inom ER och
utjämna lokala koncentrationsskillnader. Vi visar att ER i nervceller kan
genomgå snabb fission (= fragmentering) och därefter fusion. Detta fenomen
har tidigare inte... (More) - Popular Abstract in Swedish
Det endoplasmatiska retiklet (ER) är nödvändigt för proteinsyntes och
posttranslationell proteinmodifiering. ER är också iblandat i cellstress och
utgör den största intracellulära kalciumkällan i nervceller. Neuronalt ER bildar
ett kontinuerligt nätverk som består av dels rörliknande förbindelser (tubules)
och större cisterner. Att nätverket utgör en kontinuerlig lumen är en viktig
egenskap som gör att proteiner och kalciumjoner kan röra sig inom ER och
utjämna lokala koncentrationsskillnader. Vi visar att ER i nervceller kan
genomgå snabb fission (= fragmentering) och därefter fusion. Detta fenomen
har tidigare inte rapporterats i neuron. Våra fynd visar att ER-fission i
hippokampusneuron i primärkultur eller organotypa kulturer induceras av
kalciuminflöde genom en särskild glutamatreceptorklass: NMDA-receptorn.
Genom farmakologiska försök har vi visat att ER-fission induceras oberoende
av kalciumfrisättning från ER. Dessutom har vi funnit att mild hypotermi leder
till ökad fission. Detta är av intresse eftersom mild hypotermi har visat sig ha
en skyddande effekt i experimentella stroke-modeller. Slutligen har vi med 2-
fotonmikroskopi kunnat visa att snabb ER-fission också sker i hjärnan in situ i
en djurmodell av hjärtstopp. Vi har studerat ER struktur med ljusmikroskopi
(konfokal och 2-foton) i levande nervceller i primärkultur och organotypa
kulturer samt i den levande hjärnan. För odlade celler användes transfektion
för att få dessa celler att uttrycka fluorescerande proteiner specifikt i ER. För
imaging av organotypa kulturer och hjärnan in situ genererades olika transgena
musstammar med expression av ER-markörerna i olika populationer av
neuron. Dessutom har transmissionselektronmikroskopi använts på fixerad
vävnad från organotypa kulturer. För att möjliggöra kvantifiering av ER
fission och fusion har vi utvecklat en ny dataanalysmetod baserad på
”fluorescence recovery after photobleaching” (FRAP). Våra data visar att ER
är en dynamisk modell och vi föreslår en modell för ER-struktur där
kontinuiteten av ER hela tiden bestäms av en jämvikt mellan fission och
fusion. Aktivering av NMDA-receptorn skiftar jämvikten mot fission medan
inhibering av NMDA-receptorn har motsatt effekt. Denna hittills okända
strukturella dynamik har sannolikt viktiga funktionella konsekvenser för såväl
fysiologiska som patofysiologiska processer vilket diskuteras i denna
avhandling. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1730898
- author
- Kucharz, Krzysztof LU
- supervisor
- opponent
-
- Professor Hanse, Eric, University of Gothenburg, Dept. of Physiology/Neuro
- organization
- publishing date
- 2010
- type
- Thesis
- publication status
- published
- subject
- keywords
- fragmentation, fission, endoplasmic reticulum, neurons, FRAP, primary cultures, organotypic slice cultures, in vivo, in situ, calcium, fusion, NMDA receptor, 2-photon microscopy, confocal microscopy
- in
- Lund University Faculty of Medicine Doctoral Dissertation Series
- volume
- 2010:129
- pages
- 106 pages
- publisher
- Department of Clinical Sciences, Lund University
- defense location
- Segerfalksalen, Wallenberg Neuroscience Centre, Lund, Sweden.
- defense date
- 2010-12-13 09:00:00
- ISSN
- 1652-8220
- ISBN
- 978-91-86671-45-7
- language
- English
- LU publication?
- yes
- additional info
- The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Laboratory for Experimental Brain Research (013041000)
- id
- 60f5e176-9ed2-4916-89c5-523d8a6dd2cd (old id 1730898)
- date added to LUP
- 2016-04-01 14:11:30
- date last changed
- 2020-09-16 16:14:18
@phdthesis{60f5e176-9ed2-4916-89c5-523d8a6dd2cd,
abstract = {{The endoplasmic reticulum (ER) is crucial for protein synthesis and protein<br/><br>
maturation, is involved in cell stress and serves in neurons as the major<br/><br>
intracellular Ca2+ store. Neuronal ER forms a continuous network of cisterns<br/><br>
and tubules extending from soma to a subset of dendritic spines. The continuity<br/><br>
of ER structure is important for maintaining ER basic functions and necessary<br/><br>
for proteins and ions to diffuse and equilibrate within its lumen.<br/><br>
We show that ER in neurons can undergo rapid fission (=fragmentation) and<br/><br>
subsequently fusion. This phenomenon was previously unknown in neurons.<br/><br>
Our findings show that ER fission is induced during N-methyl D-aspartate<br/><br>
(NMDA) receptor-mediated Ca2+ entry to the cell in murine primary cultures<br/><br>
and hippocampal slice cultures. Using different pharmacological approaches,<br/><br>
we demonstrate, that ER fission is triggered independently on Ca2+ from ER<br/><br>
stores. Subsequently, we show that mild hypothermia, reported to be protective<br/><br>
in experimental stroke models, enhances ER fragmentation. Finally, we<br/><br>
validate the occurrence of rapid neuronal ER fission in an animal cardiac arrest<br/><br>
model of cerebral ischemia.<br/><br>
We assessed ER structure using confocal microscopy live cell and tissue<br/><br>
imaging, 2-photon laser scanning microscopy and transmission electron<br/><br>
microscopy (TEM). The fluorescence imaging was performed on murine<br/><br>
primary cultures cotransfected to express cytosolic and ER-specific markers;<br/><br>
hippocampal slices from transgenic mice expressing ER-specific marker; as<br/><br>
well as in transgenic living animals. To characterize the fission-fusion in a<br/><br>
quantitative way, we developed a new data analysis method based on<br/><br>
fluorescence recovery after photobleaching (FRAP).<br/><br>
Our data show that neuronal ER is a dynamic organelle. We propose a model<br/><br>
of ER continuity, where ER is in equilibrium with fission-fusion events.<br/><br>
Stimulation of NMDA receptors shifts the equilibrium towards the<br/><br>
fragmentation, while inhibiting NMDA receptors promotes the continuous<br/><br>
state of ER. We conclude that ER fission-fusion may be of importance in<br/><br>
physiology and disease. The molecular machinery regulating the reversible<br/><br>
changes in ER morphology remains unknown.}},
author = {{Kucharz, Krzysztof}},
isbn = {{978-91-86671-45-7}},
issn = {{1652-8220}},
keywords = {{fragmentation; fission; endoplasmic reticulum; neurons; FRAP; primary cultures; organotypic slice cultures; in vivo; in situ; calcium; fusion; NMDA receptor; 2-photon microscopy; confocal microscopy}},
language = {{eng}},
publisher = {{Department of Clinical Sciences, Lund University}},
school = {{Lund University}},
series = {{Lund University Faculty of Medicine Doctoral Dissertation Series}},
title = {{Endoplasmic Reticulum Dynamic Structural Changes in Neurons: The Fission-Fusion Phenomena}},
url = {{https://lup.lub.lu.se/search/files/3835472/1730899.pdf}},
volume = {{2010:129}},
year = {{2010}},
}