The impact of sequence variation on acid sphingomyelinase activity in the context of major depressive disorder [Elektronische Ressource] = Der Einfluss von Sequenzvariationen auf die Aktivität der sauren Sphingomyelinase im Kontext der majoren Depression / vorgelegt von Cosima Rhein

De
The impact of sequence variation on Acid Sphingomyelinase activity in the context of Major Depressive Disorder Der Einfluss von Sequenzvariationen auf die Aktivität der Sauren Sphingomyelinase im Kontext der Majoren Depression Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat. vorgelegt von Cosima Rhein aus Fürth Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 23. Mai 2011 Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink Erstberichterstatter: Prof. Dr. Uwe Sonnewald Zweitberichterstatter: Prof. Dr. Johannes Kornhuber ἔνθ᾽ αὖτ᾽ ἄλλ᾽ ἐνόησ᾽ Ἑλένη ιὸς ἐκγεγαυῖα αὐτίκ᾽ ἄρ᾽ εἰς οἶνον βάλε φάρακον, ἔνθεν ἔπινον, νηπενθές τ᾽ ἄχολόν τε, κακῶν ἐπίληθον ἁπάντων. Da entsann die zeusentsprossene Helena andres. Und sie warf in den Wein, von welchem sie tranken, ein Mittel gegen Kummer und Groll und aller Übel Gedächtnis. Homer, Odyssee Danksagung Herrn Professor Kornhuber danke ich dafür, dass ich meine Doktorarbeit an der Psychiatrischen und Psychotherapeutischen Klinik durchführen und die ausgezeichnete Infrastruktur des Labors für molekulare Neurobiologie nutzen durfte.
Publié le : samedi 1 janvier 2011
Lecture(s) : 27
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Source : D-NB.INFO/1013100956/34
Nombre de pages : 108
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The impact of sequence variation on
Acid Sphingomyelinase activity in the context of
Major Depressive Disorder

Der Einfluss von Sequenzvariationen auf die
Aktivität der Sauren Sphingomyelinase
im Kontext der Majoren Depression




Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades Dr. rer. nat.


vorgelegt von
Cosima Rhein
aus Fürth


Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der
Friedrich-Alexander-Universität Erlangen-Nürnberg












Tag der mündlichen Prüfung: 23. Mai 2011
Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink
Erstberichterstatter: Prof. Dr. Uwe Sonnewald
Zweitberichterstatter: Prof. Dr. Johannes Kornhuber



































ἔνθ᾽ αὖτ᾽ ἄλλ᾽ ἐνόησ᾽ Ἑλένη
ιὸς ἐκγεγαυῖα
αὐτίκ᾽ ἄρ᾽ εἰς οἶνον βάλε φάρακον, ἔνθεν ἔπινον,
νηπενθές τ᾽ ἄχολόν τε, κακῶν ἐπίληθον ἁπάντων.


Da entsann die zeusentsprossene Helena andres.
Und sie warf in den Wein, von welchem sie tranken, ein Mittel
gegen Kummer und Groll und aller Übel Gedächtnis.


Homer, Odyssee

Danksagung

Herrn Professor Kornhuber danke ich dafür, dass ich meine Doktorarbeit an der
Psychiatrischen und Psychotherapeutischen Klinik durchführen und die ausgezeichnete
Infrastruktur des Labors für molekulare Neurobiologie nutzen durfte. Das interdisziplinäre
Arbeiten an der Klinik hat mir interessante Einblicke in das Feld der klinischen
Neurowissenschaften ermöglicht. Eine bereichernde Erfahrung war es auch, an verschiedenen
internationalen Kongressen teilnehmen zu dürfen – herzlichen Dank dafür.

Großer Dank gilt Herrn Professor Sonnewald für die Übernahme der Begutachtung dieser
Arbeit.

Bei Herrn Dr. Martin Reichel bedanke ich mich für die inhaltliche Betreuung dieser Arbeit,
seine Diskussionsbereitschaft und sein Vertrauen in mein experimentelles Arbeiten.
Besonders danke ich ihm für die verlässliche Zusammenarbeit.
Herrn Dr. Philipp Tripal danke ich für die praktische Betreuung dieser Arbeit, seine große
Hilfsbereitschaft und die wertvolle Unterstützung. Ich danke ihm sehr, dass er mir viele
Methoden nahe gebracht und mich für das experimentelle Arbeiten begeistert hat.

Ein großes Dankeschön geht an Frau Alice Konrad für die exzellente experimentelle
Unterstützung im letzten Jahr meiner Doktorarbeit. Ihre perfekte und weitsichtige
Arbeitsweise in sämtlichen Methoden des Labors machte es zum Vergnügen, mit ihr
zusammen zu arbeiten.
Frau Michaela Schäfer danke ich sehr für die hervorragende Unterstützung im Bereich der
Zellkultur. Der hohe Standard ihrer Arbeitsweise ermöglichte erst eine Vielzahl an
Experimenten mit verschiedensten Zelllinien.
Bei Frau Sabine Müller bedanke ich mich für die ausgezeichnete Unterstützung bei den
unzähligen Schritten des Klonierens und ihre äußerst zuverlässige Arbeitsweise.

Frau Dr. Christiane Mühle danke ich für TLC-Messungen, die stets interessanten Ideen
bezüglich des Laboralltags und die Versorgung mit Werbematerial und Sprossen.
Ein Dankeschön gilt Herrn Stefan Hofmann für TLC-Messungen und seine unterhaltsamen
Gedankenexperimente über verschiedenste Themen.
Bei Frau Dr. Angela Seebahn bedanke ich mich für die unkomplizierte und erfolgreiche
Kooperation bezüglich der Massenspektrometrie.
Frau Dr. Rotter-Neubert und Herrn Dr. Bernd Lenz danke ich für die gute Zusammenarbeit
und ihre Bereitschaft, meine Experimente jederzeit durch Blutentnahmen zu unterstützen.
Bei den Mitarbeitern der Abteilung für molekulare Neurologie bedanke ich mich für die
fruchtbare Zusammenarbeit und die kollegiale Nachbarschaft.
Allen aktuellen und bereits ausgeschiedenen Kollegen der molekularen Neurobiologie sage
ich Dankeschön dafür, dass sie die Zeit während meiner Doktorarbeit mitgestaltet und meist
bereichert haben.

Meinem Molmädels-Stammtisch danke ich sehr für die fachliche und mentale Unterstützung.
Herrn ORR Bernd W. Göppner danke ich für die umfassende Unterstützung, sein Verständnis
für alle Widrigkeiten des Forscherlebens und seine Fähigkeit, mich stets zu motivieren.
Von Herzen danke ich meinen Eltern und Schwestern für den unerschütterlichen Beistand in
allen Lebenslagen.


Contents

Contents


List of figures .................................................................................VIII
List of tables ...................................................................................... IX
List of abbreviations...........................................................................X

1. Zusammenfassung........................................................................ 12
Summary ........................................................................................... 13

2. Introduction .................................................................................. 14
2.1 Lysosomal sphingolipid disorders of the brain .......................................................14
2.1.1 Sphingolipids play diverse roles in cellular processes........................................15
2.1.1.1 Sphingolipids: Functional diversity by structural diversity....................15
2.1.1.2 Roles of sphingolipids for the CNS ........................................................16
2.1.1.3 The lipid messenger ceramide ................................................................16
2.1.2 Molecular defects of lysosomal enzymes ...........................................................18
2.1.2.1 Primary defects .......................................................................................19
2.1.2.2 Posttranslational defects..........................................................................20
2.1.2.3 Defective trafficking ...............................................................................20
2.2 Acid sphingomyelinase in health and disease..........................................................21
2.2.1 Acid Sphingomyelinase – the lysosomal enzyme for sphingomyelin breakdown
.............................................................................................................................21
2.2.1.1 Genomic organization and transcription.................................................22
2.2.1.2 Sequence variations of Acid Sphingomyelinase.....................................23
2.2.1.3 Properties of the Acid Sphingomyelinase protein ..................................23
2.2.2 Regulation of Acid Sphingomyelinase activity ..................................................25
2.2.2.1 Transcriptional level ...............................................................................25
2.2.2.2 Posttranscriptional level..........................................................................26
2.2.2.3 Posttranslational modifications...............................................................26
2.2.2.4 Regulation via degradation .....................................................................26
2.2.3 Acid Sphingomyelinase and pathologic context.................................................27
2.2.3.1 Decreased activity of Acid Sphingomyelinase .......................................27
2.2.3.2 Increased activity of Acid Sphingomyelinase.........................................28
2.3 The role of Acid Sphingomyelinase in Major Depressive Disorder ......................28
2.3.1 Results of the pilot study.....................................................................................29
2.3.2 Sequence variations of Acid Sphingomyelinase in depressed patients ..............30
2.4 Aims of the study........................................................................................................30

3. Material and Methods.................................................................. 32
3.1 Material.......................................................................................................................32
3.1.1 Enzymes..............................................................................................................32
3.1.2 Antibodies...........................................................................................................32
3.1.2.1 Primary antibodies ..................................................................................32
3.1.2.2 Secondary antibodies ..............................................................................33
3.1.3 Vectors and plasmids ..........................................................................................33
3.1.3.1 Vectors ....................................................................................................33
V Contents
3.1.3.2 Plasmids ..................................................................................................33
3.1.4 Biological material..............................................................................................35
3.1.4.1 Bacteria ...................................................................................................35
3.1.4.2 Eucaryotic cells.......................................................................................35
3.1.5 Buffers and solutions ..........................................................................................35
3.2 Methods.......................................................................................................................39
3.2.1 Cell-biological methods......................................................................................39
3.2.1.1 Cell culture..............................................................................................39
3.2.1.2 Transient expression of ASM .................................................................40
3.2.1.3 Immunocytochemistry ............................................................................40
3.2.1.4 Preparation of whole cell extracts...........................................................41
3.2.2 DNA technology .................................................................................................41
3.2.2.1 PCR for cloning approach.......................................................................41
3.2.2.2 Sub-cloning of ASM into pmCherry-N1 expression vector ...................42
3.2.2.3 Site-directed mutagenesis .......................................................................43
3.2.2.4 PCR for detection of splice variants .......................................................43
3.2.2.5 Agarose gel electrophoresis ....................................................................44
3.2.2.6 Cloning of ASM into FLAG-N2 expression vector................................45
3.2.2.7 Transformation of bacteria with DNA....................................................46
3.2.2.8 Isolation of plasmid DNA.......................................................................47
3.2.2.9 Sequencing of cDNA ..............................................................................47
3.2.3 RNA isolation and cDNA synthesis ...................................................................48
3.2.3.1 RNA isolation .........................................................................................48
3.2.3.2 Synthesis of cDNA .................................................................................49
3.2.4 Protein-biochemical analyses..............................................................................49
3.2.4.1 Determination of protein concentration..................................................49
3.2.4.2 SDS-PAGE and Western blot analysis ...................................................49
3.2.4.3 In vitro determination of ASM activity ..................................................51
3.2.4.4 MALDI-TOF MS analysis of cellular ceramide and sphingomyelin
levels .......................................................................................................52
3.2.5 In silico analyses and statistics ...........................................................................52

4. Results............................................................................................ 53
4.1 Impact of DNA sequence variations on Acid Sphingomyelinase activity .............53
4.1.1 Expression of recombinant ASM variants ..........................................................55
4.1.2 Characterization of ASM variants used for model system .................................56
4.1.2.1 Catalytic activity of ASM model system variants ..................................57
4.1.2.2 Localization of ASM model system variants..........................................58
4.1.3 Characterization of ASM variant p.P325A.........................................................60
4.1.3.1 ASM variant p.P325A is catalytically inactive.......................................61
4.1.3.2 ASM variant p.P325A localizes in lysosomes........................................61
4.1.4 Characterization of ASM variant p.A487V ........................................................63
4.1.4.1 Catalytic activity of ASM variant p.A487V is equivalent to wild type..63
4.1.4.2 ASM variant p.A487V localizes in lysosomes .......................................64
4.1.5 Characterization of ASM variant p.G506R ........................................................65
4.1.5.1 ASM variation p.G506R impacts catalytic activity ................................65
4.1.5.2 ASM variation p.G506R impacts localization........................................66
4.1.6 Summary: Characterization of ASM variants.....................................................69
4.2 Impact of alternative splicing on Acid Sphingomyelinase activity........................70
4.2.1 Structure of new ASM splice variants ................................................................73
4.2.2 New splicing motifs are conserved in primates ..................................................74
VI Contents
4.2.3 Biochemical characterization of new ASM splice variants ................................74
4.2.3.1 New ASM splice variants are expressed in a cell culture model............77
4.2.3.2 New ASM splice variants are catalytically inactive in vitro...................77
4.2.3.3 New ASM splice variants are catalytically inactive in vivo ...................78
4.2.4 New ASM splice variants localize within different subcellular compartments..79
4.2.5 New ASM splice variants show differential expression patterns in human blood
cells .....................................................................................................................82
4.2.5.1 New ASM splice variants are detected specifically by RT-PCR............82
4.2.5.2 New ASM splice variants are differentially expressed in healthy
volunteers................................................................................................83
4.2.5.3 New ASM splice variants are differentially expressed in depressed
patients before and after antidepressant therapy.....................................84
4.2.6 New splice variant ASM-5 exerts a dominant-negative effect on full-length Acid
Sphingomyelinase...............................................................................................86
4.2.6.1 Upon serum starvation ASM-5 localizes within lysosomes ...................86
4.2.6.2 ASM-5 is a dominant-negative ASM isoform........................................88

5. Discussion ...................................................................................... 90
5.1 DNA sequence variations impact Acid Sphingomyelinase activity .......................90
5.1.1 The C-terminal domain harbours essential parts for activation and secretion of
Acid Sphingomyelinase ......................................................................................91
5.1.2 Processing of Acid Sphingomyelinase is essential for its regulation in pathologic
context.................................................................................................................92
5.2 Alternative splicing impacts Acid Sphingomyelinase activity ...............................93
5.2.1 Alternative splicing of Acid Sphingomyelinase could predominate in brain and
blood cells ...........................................................................................................94
5.2.2 Acid Sphingomyelinase displays a defined set of conserved splicing motifs ....94
5.2.3 Acid Sphingomyelinase activity is regulated by alternative splicing .................96

6. Bibliography.................................................................................. 98
7. Appendix ..................................................................................... 107
7.1 Chemicals..................................................................................................................107
7.2 Technical device .......................................................................................................108
7.3 Auxiliary material....................................................................................................108


VII List of figures
List of figures

Fig. 2.1 The ‘ceramide / sphingosine-1-phosphate rheostat’ concept.................................17
Fig. 2.2 ASM is subject to alternative splicing events........................................................23
Fig. 2.3 ASM is composed of several domains...................................................................24
Fig. 4.1 Position of ASM variations investigated in this study...........................................53
Fig. 4.2 Cloning approach for the generation of recombinant ASM constructs containing
different ASM variations and FLAG- or Cherry-epitope ......................................54
Fig. 4.3 All investigated ASM variants are translated into protein and display
predicted sizes........................................................................................................55
Fig. 4.4 ASM variants were transiently expressed to a different extent .............................56
Fig. 4.5 ASM model system variants display predicted enzymatic activity levels.............58
Fig. 4.6 Subcellular localization patterns of ASM model system variants ASM wild type
and ASM variants p.S508A and p.M381I..............................................................59
Fig. 4.7 Co-localization of ASM model system variants with subcellular organelles........60
Fig. 4.8 ASM variant p.P325A does not exert catalytic activity compared to ASM
wild type.................................................................................................................61
Fig. 4.9 ASM variant p.P325A localizes in a dotted subcellular pattern ............................62
Fig. 4.10 ASM variant p.P325A is localized within lysosomes............................................62
Fig. 4.11 Enzymatic activities of ASM variant p.A487V and ASM wild type reach similar
levels ......................................................................................................................63
Fig. 4.12 ASM variant p.A487V localizes in a dotted subcellular pattern ...........................64
Fig. 4.13 ASM variant p.A487V localizes within lysosomes ...............................................64
Fig. 4.14 ASM variation p.G506R causes a decrease in enzymatic activity.........................66
Fig. 4.15 Subcellular localization patterns of ASM variants containing the variation
p.G506R.................................................................................................................67
Fig. 4.16 Co-localization of ASM variants containing variation p.G506R with subcellular
organelles. ..............................................................................................................68
Fig. 4.17 Coding sequences of full-length ASM and new alternatively-spliced transcripts
differ locally...........................................................................................................72
Fig. 4.18 Schematic structure of new splice variants ASM-5, -6 and -7 ..............................73
Fig. 4.19 Splicing patterns of novel transcripts ASM-6 and -7 are conserved in primates...74
Fig. 4.20 Novel variants ASM-5 to -7 differ regarding their putative protein sequence and
protein domain structure ........................................................................................76
Fig. 4.21 Expression of ASM-FLAG constructs in HeLa cells ............................................77
Fig. 4.22 Novel variants ASM -5 to -7 are catalytically inactive..........................................78
Fig. 4.23 New ASM splice variants modulate ceramide to sphingomyelin ratios................79
Fig. 4.24 Subcellular localization patterns of ASM-1 and new ASM splice variants...........80
Fig. 4.25 Co-localization of new ASM splice variants with subcellular organelles. ............81
Fig. 4.26 Designed oligonucleotides serve for specific amplification of new ASM splice
variants...................................................................................................................83
Fig. 4.27 Expression patterns of ASM splice variants vary in human blood cells................84
Fig. 4.28 Expression patterns of ASM splice variants vary in blood cells of depressed
patients before and after administering of antidepressants....................................85
Fig. 4.29 Subcellular localization pattern of ASM-5 upon serum starvation........................87
Fig. 4.30 ASM-5 localizes within lysosomes upon serum starvation ...................................87
Fig. 4.31 ASM-5 exerts a dominant-negative effect on ASM-1 upon serum starvation.......89

VIII List of tables
List of tables

Tab. 3.1 Primary antibodies in cotext of their application...................................................32
Tab. 3.2 Secondary antibodies in cotext of their application...............................................33
Tab. 3.3 Vectors used in this study ......................................................................................33
Tab. 3.4 Plasmids used in this study ....................................................................................34
Tab. 3.5 Oligonucleotides used for cloning of ASM into pSC-B vector and FLAG-N2
expression vector ...................................................................................................42
Tab. 3.6 Oligonucleotides used for cloning of ASM from FLAG-N2 vector into
pmCherry-N1 vector. .............................................................................................42
Tab. 3.7 Oligonucleotides used for site-directed mutagenesis of ASM wild type-FLAG
construct to generate different ASM variants ........................................................43
Tab. 3.8 Oligonucleotides used for amplification of ASM splice variants..........................44
Tab. 3.9 Oligonucleotides used for sequencing of ASM plasmids......................................47
Tab. 4.1 ASM variants display differential properties.........................................................69

IX List of abbreviations
List of abbreviations

AA amino acid
AC Acid Ceramidase
APS ammonium persulfate
ASM Acid Sphingomyelinase
BES 2-hydroxyethyl-2-aminoethanesulfonic acid
bp base pairs
BSA bovine serum albumine
-2
c centi (10 )
°C degree Celsius
C-terminal carboxy-terminal
CNS central nervous system
cDNA coding DNA
DMEM Dulbecco’s Modified Eagle’s Medium
DMSO dimethylsulfoxide
DMF 2,5-dimethylfurane
DNA deoxyribonucleic acid
dNTP deoxyribonucleoside-5’-triphosphate
DTT dithiotreitol
EC enzyme classification number
ECL enhanced chemiluminescence
E. coli Escherichia coli
EDTA ethylenediaminetetraacetate
ER endoplasmic reticulum
FCS fetal calf serum
Fig. figure
fw forward
g grams
g gravitation constant
GAPDH glycerinaldehyd-3-phosphat-dehydrogenase
GFP green fluorescent protein
h hour(s)
H4 neuroglioma cell line
H O aqua bidestillata 2 bidest
HPRT hypoxanthine-phosphoribosyl transferase
kb kilo base pairs
kDa kilo daltons
l litre
LAMP1 lysosomal-associated membrane protein 1
LB Luria Bertani Medium
LPS lipopolysaccharide
M molar
-3
m milli (10 )
-6
μ micro (10 )
MDD major depressive disorder
mRNA messenger ribonucleic acid
min minute(s)
MALDI-TOF MS matrix assisted laser desorption/ionisation time of flight
mass spectrometry
MCS multiple cloning site
X

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