dc.contributor.author
Das, Debashish
dc.date.accessioned
2018-06-07T19:07:31Z
dc.date.available
2005-03-30T00:00:00.649Z
dc.identifier.uri
https://refubium.fu-berlin.de/handle/fub188/5768
dc.identifier.uri
http://dx.doi.org/10.17169/refubium-9967
dc.description
Title Page, Table of Contents
Acknowledgements 4
Abbreviations and Acronyms 6
Summary 8
Zusammenfassung 10
A. Introduction
1\. Molecular Analysis of Synaptogenesis 13
2\. Activity-independent Guidance for Synaptogenesis 14
3\. Neuronal Activity Role in Synaptogenesis 15
3.1 Neuromuscular junction 16
3.2 Visual pathway 18
4\. Activity-dependent Gene and Protein Regulation 19
4.1 Gene regulation 20
4.2 Protein regulation 21
B. Aim and Hypothesis 23
1\. PART-I: Is CALEB an activity-dependent regulated molecule 23
1.1 Hypothesis: 23
1.2 Objective: 23
2\. PART-II: What is the mechanism of the activity-dependent down regulation
23
2.1 Hypothesis: 23
2.2 Objective: 24
3\. PART-III: Is the ectodomain shedding of CALEB caused by a protease 24
3.1 Hypothesis: 24
3.2 Objective: 24
C. Materials and Methods 25
1\. MATERIALS 25
1.1 Chemicals, medium and enzymes 25
1.2 Consumables and special appliances 25
1.3 Softwares used 26
1.4 Antibodies 26
1.5 cDNAs 27
1.6 Competent cells (Bacteria) 27
1.7 Animals, cells (primary and cell lines), tissues 28
2\. METHODS 28
2.1 Cell culture methods 28
2.1.1 Maintenance of cell line 28
2.1.2 Primary cell culture 29
2.2 Biochemical methods 30
2.2.1 Electrophoresis and Western blot 30
2.2.2 Quantification of Western blots 32
2.2.3 Deglycosylation of tissue 32
2.2.4 Protein extraction from cells and tissue 33
2.2.5 Biotinylation, avidin precipitation and elution 33
2.2.6 Biotin Internalization Assay 35
2.2.7 Identification of a released CALEB fragment 36
2.2.8 Characterization of membrane associated protease that cleaves CALEB 36
2.2.9 In-vitro CALEB cleavage reaction 36
2.3 Molecular biological methods 37
2.3.1 Preparation of competent cells 37
2.3.2 Transformation 38
2.3.3 Plasmid DNA preparation 38
2.3.4 Restriction enzyme digestion 38
2.3.5 Transfection of cDNAs to cell lines 39
2.4 Histological methods 39
2.4.1 Tissue sectioning 39
2.4.2 Neurohistology staining 40
2.4.3 Immunohistochemical staining 40
2.5 Immunocytochemistry 41
2.5.1 Fixation and immunostaining 41
2.6 Post-synaptic density (PSD) preparation 42
2.6.1 Animals and reagents 42
2.6.2 Protocol for PSD preparation 42
2.7 Superior colliculus incubation experiments 44
D. RESULTS 46
I Neuronal activity dependent regulation of CALEB 46
1\. Activity dependent down regulation of cell surface CALEB 46
1.1 Characterization of CALEB in E11 chick retina 46
1.2 Cell surface CALEB down regulation with neuronal activity 49
1.3 Cell surface down regulation occurs with the activation of glutamate
receptors 51
1.4 Down regulation of CALEB is an action potential independent process 53
2\. Role of Calcium in cell surface down regulation of CALEB 54
2.1 Calcium influx is necessary for the down regulation of CALEB 54
2.2 Down regulation is a calmodulin and calcineurin dependent process 56
3\. Shedding is the cause of down regulation of cell surface CALEB 59
3.1 Down regulation of cell surface CALEB is an outcome of ectodomain shedding
59
3.2 Presence of the shed ectodomain fragment of CALEB protein 61
3.4 Presence of the membrane attached part of CALEB after shedding 62
3.5 Presence of lower fragment in the developmental stages of chick 64
4\. Metalloprotease inhibition, inhibits shedding 66
4.1 Shedding of the ectodomain occurs by membrane associated proteases 67
4.2 ADAM group of metalloproteases is responsible for shedding 68
4.3 ADAM 10 and ADAM 17 are responsible for causing the ectodomain cleavage of
CALEB 70
5\. Shedding of CALEB in the presence of pharmacological reagents that affect
intracellular signalling pathways 72
5.1 Involvement of Erk kinase pathway in shedding of CALEB ectodomain 73
5.2 Shedding is independent of protein synthesis 74
6\. Calcium dependent upregulation of total CALEB 75
6.1 Total CALEB protein upregulation upon neuronal activity 76
6.2 Total CALEB increased because of more translation 77
II. Characterization of CALEB in mouse neural tissue 79
1\. Expression of mouse CALEB 79
1.1. Tissue specific expression of mouse CALEB 79
1.2 Mouse CALEB enrichment in the synaptic junctions 80
2\. Developmental expression profile of mouse CALEB 81
2.1 Superior colliculus 81
2.2 Mouse Retina 83
3\. Activity-dependent regulation of mouse CALEB in superior colliculus 85
E. Discussion 86
1\. Characterization and Distribution of CALEB 86
2\. Activity dependent down Regulation of CALEB 88
3\. CALEB down regulation is a Calcium dependent process 91
4\. Down regulation as a consequence of CALEB ectodomain shedding 94
5\. Metaloproteases cause the ectodomain shedding in the chick retina cultures
98
7\. Upregulation of chick CALEB by neuronal activity 101
8\. Mouse CALEB expression and regulation by activity 102
F. Future prospects 104
G. LITERATURE 105
H. Appendix 121
I. Erklärung 122
J. Curriculum Vitae 123
dc.description.abstract
Synaptogenesis is a collaborative effort of activity-independent processes
specifying neuronal connections, and activity-dependent processes refining
these initial synaptic connections. CALEB, an activity-dependent modulated
protein, might be implicated in this refinement. The aim of my work was to
investigate the mechanism of CALEB (Chicken Acidic Leucine rich EGF-like
domain containing brain protein) down regulation, which was identified in an
initial screen. I showed that cell surface CALEB down regulation was enhanced
in retinal cultures incubated with KCl or agonist of glutamate receptors.
Modulation of extracellular calcium concentration and experiments using
blockers of calmodulin revealed the involvement of calcium in the mechanism of
down regulation. Since CALEB belongs to the EGF family of growth and
differentiation factors that undergo cell surface cleavage, I hypothesized a
similar phenomenon responsible for the cell surface down regulation of CALEB.
Isolation of a soluble component in supernatants of KCl treated cultures and a
remaining membrane-tethered part in KCl incubated retinal cultures confirmed
my hypothesis. I showed in membrane fraction incubation experiments that
cleavage of CALEB ectodomain was caused by the catalytic action of a membrane
protease, which could be prevented in the presence of metalloprotease
inhibitor. For a detailed analysis of CALEB shedding, I showed that blockade
of two ADAMs (A disintegrin and metalloprotease) with inhibitors as well as
increased expression of one ADAM resulted in a decrease of total CALEB in
comparison to cell lines co-expressing dominant negative form of the same ADAM
and CALEB. Further experiments showed the involvement of Erk kinase in
ectodomain shedding of CALEB. In another part of the project, I investigated
the expression and localization of CALEB in the visual system in order to
predict a plausible role of CALEB during development. Deglycosylation
experiments showed CALEB as a highly glycosylated brain specific protein.
Subcellularly, CALEB was enriched in the synaptic junction fractions pointing
to a possible role of CALEB in synapses. The developmentally regulated
expression of CALEB was observed in the visual system. My findings suggest
that neuronal activity induced by depolarization facilitates ectodomain
shedding of CALEB by the action of metalloproteases. The expression profile
and localization confirms CALEB to be a brain specific, glycosylated
transmembrane protein, which might have a role during the formation or
maintenance of synapses.
de
dc.description.abstract
Die Bildung von Synapsen wird durch das Zusammenspiel von aktivitäts-
unabhängigen Prozessen, die neuronale Verbindungen spezifizieren, und von
aktivitäts-abhängig Prozessen, welche die Feinabstimmung synaptischer
Verbindungen beeinflussen, bestimmt. Bei Letzterem könnte CALEB, ein
aktivitat-abhängig moduliertes Protein, von Bedeutung sein. Das Ziel meiner
Arbeit war es, den Mechanismus der aktivitäts-abhängigen Modulation von CALEB
(Chicken Acidic Leucine-rich EGF-like domain containing brain protein) zu
untersuchen. Ich konnte zeigen, dass in Retinakulturen nach Inkubation mit KCl
oder Glutamatrezeptoragonisten eine Herunterregulierung von CALEB an der
Zelloberfläche innerhalb von fünf Minuten verstärkt wird. Durch Änderung der
extrazellulären Kalziumkonzentration und durch die Verwendung von
Calmodulininhibitoren konnte die Beteiligung von Kalzium an diesem Prozess
demonstriert werden. Da CALEB ein Mitglied der EGF-Familie von Wachstums- und
Differenzierungsfaktoren ist, postulierte ich, dass es bei der
Herunterregulierung von CALEB zu einer Ektodomänenabspaltung kommt. Das
Auffinden einer löslichen Komponente im Überstand von KCl-behandelten
Kulturen, sowie eines membranständigen Restfragments in Retinakulturen
bestätigten meine Hypothese. Mittels Inkubation von Membranfraktionen konnte
ich zeigen, dass die Abspaltung der CALEB-Ectodomäne durch
Metalloproteaseinhibitoren verhindert wird. Zur genaueren Charakterisierung
der Abspaltung der CALEB-Ectodomäne wurden die beiden ADAM Proteasen (A
disintegrin and metalloprotease), ADAM 10 und ADAM 17 untersucht. Die
Blockierung von ADAM 10 und ADAM 17 mit pharmakologischen Inhibitoren
verhinderte die Spaltung von CALEB. Darüberhinaus führte eine erhöhte
Expression von ADAM 10 zu einer Abnahme des CALEB Gesamtproteins im Vergleich
zu dominant negatives ADAM 10. Weitere pharmakologischen Experimente ließen
auf eine Beteiligung der Erk-Kinase bei der Abspaltung der CALEB-Ectodomäne
schliessen. Eine weitere Aufgabe dieser Arbeit war es, die Expression und
Lokalisation von CALEB im visuellen System zu untersuchen.
Deglykosylierungsexperimente zeigten, dass es sich bei CALEB um ein in hohem
Maße glykosyliertes, Gehirn-spezifisches Protein handelt. Subzellulär wird
CALEB mittels "post-synaptic density preparation" in der "synaptischen
junction Fraktion" angereichert, was auf eine mögliche Rolle von CALEB in
Synapsen hindeutet. Eine entwicklungs-abhängig regulierte Expression von CALEB
wurde im visuellen System beobachtet. Meine Untersuchungen lassen vermuten,
dass neuronale Aktivität die Abspaltung der äußeren Domäne durch
Metalloproteasen fördert. Das Expressionsprofil und die Lokalisation von CALEB
bestätigen, dass es sich bei CALEB um ein gehirnspezifisches, glycosyliertes
Transmembranprotein handelt, welchem eine Rolle während der Bildung und
Erhaltung von Synapsen zukommen könnte.
de
dc.rights.uri
http://www.fu-berlin.de/sites/refubium/rechtliches/Nutzungsbedingungen
dc.subject
activity-dependent
dc.subject
ectodomain shedding
dc.subject
metalloprotease
dc.subject.ddc
500 Naturwissenschaften und Mathematik::540 Chemie::540 Chemie und zugeordnete Wissenschaften
dc.title
CALEB, an activity-dependent down regulated molecule
dc.contributor.firstReferee
Prof. Dr. Fritz G. Rathjen
dc.contributor.furtherReferee
Prof. Dr. Dietmar Kuhl
dc.date.accepted
2005-03-21
dc.date.embargoEnd
2005-04-01
dc.identifier.urn
urn:nbn:de:kobv:188-2005000807
dc.title.translated
CALEB, ein aktivitäts-abhängiges herunterreguliertes Molekül
de
refubium.affiliation
Biologie, Chemie, Pharmazie
de
refubium.mycore.fudocsId
FUDISS_thesis_000000001602
refubium.mycore.transfer
http://www.diss.fu-berlin.de/2005/80/
refubium.mycore.derivateId
FUDISS_derivate_000000001602
dcterms.accessRights.dnb
free
dcterms.accessRights.openaire
open access
