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

Kucharz, Krzysztof LU (2010) In Lund University, Faculty of Medicine Doctoral Dissertation Series 2010:129.
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)
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)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Hanse, Eric, University of Gothenburg, Dept. of Physiology/Neuro
organization
publishing date
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
ISSN
1652-8220
ISBN
978-91-86671-45-7
language
English
LU publication?
yes
id
60f5e176-9ed2-4916-89c5-523d8a6dd2cd (old id 1730898)
date added to LUP
2010-11-29 12:56:30
date last changed
2018-05-29 10:58:21
@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},
  keyword      = {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},
  pages        = {106},
  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},
  volume       = {2010:129},
  year         = {2010},
}