Regulation of Alzheimer's disease-relevant protein processing in human neurons of the LUHMES ...

167 Pages·2011·14.49 MB·English

by  Diana Scholz

Regulation of Alzheimer’s disease-relevant
protein processing in human neurons
of the LUHMES cell line

Dissertation

zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften (Dr. rer. nat.)

vorgelegt von

Diana Scholz

an der

Mathematisch-Naturwissenschaftliche Sektion
Fachbereich Biologie

Tag der mündlichen Prüfung: 28.11.2011
1. Referent: Prof. Dr. Marcel Leist
2. Referent: Prof. Dr. Christof Hauck

List of publications

Publications, integrated in this thesis:

Chapter C
Scholz, D., Pöltl, D., Genewsky, A., Weng, M., Waldmann, T., Schildknecht, S., Leist, M.
2011. Rapid, complete and large-scale generation of post-mitotic neurons from the human
LUHMES cell line. J Neurochem 119, 957-971

Chapter D
Scholz, D., Leist, M. 2011. Control of Aβ release from human neurons by differentiation
status and RET signaling. Submitted

Chapter E
Scholz, D., Leist, M. 2011. Inverse effects of BACE levels on Aβ secretion by human
neurons. Submitted

Publications, not integrated in this thesis:

Schildknecht, S., Pöltl, D., Nagel, D.M., Matt, F., Scholz, D., Lotharius, J., Schmieg, N.,
Salvo-Vargas, A., Leist, M. 2009. Requirement of a dopaminergic neuronal phenotype for
toxicity of low concentrations of 1-methyl-4-phenylpyridinium to human cells. Toxicol Appl
Pharmacol 241, 23-35

Waldmann, T., Weng, M., Zimmer, B., Pöltl, D., Scholz, D., Broeg, M., Kadereit, S.,
Wuellner, U., Leist, M. Extensive transcriptional regulation of chromatin modifiers during
human neurodevelopment. Submitted
Posters

Scholz, D., Leist, M. Characterization of pathomechanisms relevant to Alzheimer’s Disease
in a human neuronal model system. VII World Congress on Alternatives & Animal use in
the Life Sciences 2009, Rome, Italy.

Scholz, D., Leist, M. Development of a human neuronal model system for the in vitro
characterization of pathomechanisms relevant to Alzheimer’s disease. International
Conference on Alzheimer's Disease (ICAD) 2010, Honolulu, Hawai’i.

Scholz, D., Leist, M. Effects of GDNF and overexpressed BACE on APP processing in
LUHMES cells as human neuronal model system for Alzheimer’s disease. AD/PD
International Conference 2011, Barcelona, Spain.
Table of contents

Table of contents

A SUMMARY .......................................................................................................1
ZUSAMMENFASSUNG....................................................................................2

B GENERAL INTRODUCTION............................................................................3
1 From normal aging to Alzheimer’s disease (AD) .........................................3
2 Clinical symptoms and different forms of AD..............................................3
3 Histopathological features of AD: plaques and tangles..............................4
4 Aβ generation and its key players.................................................................7
4.1 Clearance of Aβ................................................................................................8
4.2 APP...................................................................................................................9
4.3 α-secretase.....................................................................................................10
4.4 BACE..............................................................................................................10
4.5 γ-secretase......................................................................................................12
5 Therapeutic aspects.....................................................................................13
5.1 BACE inhibitors...............................................................................................13
5.2 γ-secretase inhibitors ......................................................................................14
6 Model systems for AD research ..................................................................15
7 The neuronal model system used in this thesis: LUHMES.......................16
7.1 The ventral mesencephalon............................................................................17
7.2 The growth factor for proliferating LUHMES: bFGF........................................18
7.3 The growth factor for differentiating LUHMES: GDNF.....................................18
7.4 Second messenger supply from outside: db-cAMP ........................................19
8 Aims of this thesis........................................................................................20

C RAPID, COMPLETE AND LARGE-SCALE GENERATION OF POST-
MITOTIC NEURONS FROM THE HUMAN LUHMES CELL LINE.................21
1 Abstract.........................................................................................................22
2 Introduction...................................................................................................23
3 Materials and Methods.................................................................................25
4 Results...........................................................................................................30
4.1 Conversion of undifferentiated LUHMES into post-mitotic neuronal cells.......30
4.2 Electrophysiological properties of post-mitotic LUHMES................................31
4.3 Differential changes in phenotypic markers of neuronal maturation................33
4.4 Neurodevelopmental aspects of neurite growth..............................................35
4.5 Progress along the dopaminergic lineage during LUHMES maturation..........37
4.6 Robust predetermination of the neuronal fate under altered
differentiation conditions.................................................................................38
5 Discussion.....................................................................................................42
Table of contents

6 Acknowledgments........................................................................................45
7 Supplementary figures.................................................................................46

D CONTROL OF Aβ RELEASE FROM HUMAN NEURONS BY
DIFFERENTIATION STATUS AND RET SIGNALING...................................53
1 Abstract.........................................................................................................54
2 Introduction...................................................................................................55
3 Materials and Methods.................................................................................57
4 Results...........................................................................................................61
4.1 Expression and maturation of AD-relevant markers during differentiation......61
4.2 Modulation of proteolytic processing of APP in differentiated LUHMES..........62
4.3 Increased Aβ production in aged LUHMES is independent of tau changes....64
4.4 Enhanced APP processing and RET expression in the presence of GDNF....65
4.5 Triggering of increased Aβ in mature LUHMES by RET-activating ligands.....68
4.6 Immediate trigger of Aβ secretion by RET-signaling factors...........................69
4.7 Identification of the PI3K/AKT/mTOR pathway as contributor to
GDNF-triggered Aβ increase...........................................................................71
5 Discussion.....................................................................................................73
6 Acknowledgments........................................................................................76
7 Supplementary figures.................................................................................76

E INVERSE EFFECTS OF BACE LEVELS ON Aβ SECRETION
BY HUMAN NEURONS..................................................................................79
1 Abstract.........................................................................................................80
2 Introduction...................................................................................................81
3 Materials and Methods.................................................................................83
4 Results...........................................................................................................88
4.1 Neuronal differentiation and BACE overexpression of BLUHMES cells..........88
4.2 BACE expression during differentiation of BLUHMES cells............................89
4.3 BACE localization and maturation in differentiated BLUHMES.......................91
4.4 APP processing in dependence on BACE levels during differentiation...........93
4.5 Modification of Aβ generation in BLUHMES vs. LUHMES: BACE ..................94
4.6 Modification of Aβ generation in BLUHMES vs. LUHMES: α-/γ-secretase......96
4.7 Biological modulation of the Aβ effect and independence
of the BLUHMES differentiation state..............................................................98
5 Discussion...................................................................................................100
6 Acknowledgments......................................................................................103

F GENERAL DISCUSSION.............................................................................105
1 Assessment of LUHMES as neuronal model system...............................105
2 Assessment of LUHMES as system for AD-related research .................107
3 Aβ generation in dependence on GDNF-mediated RET signaling..........111
Table of contents

3.1 RET as primary culprit and mTOR as downstream confederate...................111
3.2 Implications for AD research.........................................................................113
4 Inverse relationship between BACE and Aβ levels..................................114
4.1 The proposal of a hypothetical model...........................................................114
4.2 Implications for AD research.........................................................................118
5 Conclusions ................................................................................................118

G BIBLIOGRAPHY ..........................................................................................119

H APPENDIX....................................................................................................155
1 Frequent abbreviations..............................................................................155
2 Plasmid maps..............................................................................................156

RECORD OF CONTRIBUTIONS..................................................................157

A SUMMARY
Alzheimer’s disease (AD) is a neurodegenerative disorder, whose major pathologies are the
excessive generation of amyloid β (Aβ) peptides and the aberrant phosphorylation of tau
proteins. AD exists most commonly as sporadic form (SAD) without hereditary background
(SAD). Interestingly, the level and/or activity of BACE, an enzyme crucially involved in Aβ
generation, appears to be elevated in SAD brains. In order to replicate the disease states,
transgenic mouse models and cell lines have been generated. Yet, the transfer of results
from animals to humans is difficult and cell lines often lack neuronal properties or exhibit only
weak endogenous expression of key proteins. As new approach in the framework of this
doctoral thesis, we used human neurons, differentiated in vitro from the LUHMES cell line.
Initially, we established a new 2-step differentiation protocol. We showed that by this
procedure, the precursor cells irreversibly converted into post-mitotic neurons within 5 days,
accompanied by an extensive outgrowth of neurites and the upregulation of synaptic
proteins. It was possible to trigger neuronal differentiation even in the absence of the medium
factors cAMP and GDNF, but the expression of some dopaminergic markers and the
production of dopamine depended on the presence of cAMP. Thereby, we described for the
first time LUHMES with a ‘non-dopaminergic’ phenotype, which are suitable for applications
in various research fields. Next, we characterized LUHMES as human model for AD-relevant
studies. We investigated how the expression and interaction of key proteins like amyloid
precursor protein (APP) and BACE were regulated. During long-term cell culture (10 days),
we observed that Aβ generation and tau phosphorylation continuously increased, and that
both could be pharmacologically or biologically modulated like in primary neurons. We also
revealed that the Aβ increase was induced through activation of the RET receptor by growth
factors such as GDNF, and that the PI3K pathway downstream of RET was involved. These
data indicate that a growth factor elevation, e.g. as defense mechanism in the AD brain, can
lead to further augmentation of Aβ production. Finally, we established a stable BACE-
overexpressing LUHMES cell line in order to mimic SAD conditions. Interestingly, BACE
overexpression was not constitutive but progressively increased during differentiation of the
cells, resulting in a correspondingly enhanced β-cleavage of APP. However, while moderate
BACE overexpression was linked to strong Aβ production as expected, BACE levels above a
certain threshold prompted the cells to generate significantly less Aβ than wildtype LUHMES.
In addition, the reduction of BACE activity in these cells, e.g. by treatment with low
concentrations of BACE inhibitors, led to a strong rise in Aβ. These findings contribute to the
understanding of the complex biology of APP processing and may have implications for the
development of partial BACE inhibitors as AD therapeutic.

1
A ZUSAMMENFASSUNG
Morbus Alzheimer (MA) ist eine neurodegenerative Erkrankung, die mit übermäßiger
Generierung von Amyloid β (Aβ) Peptiden und abnormaler Phosphorylierung von Tau
Proteinen einhergeht. MA kommt am häufigsten als nicht erblich bedingte, sporadische
Variante vor (SMA). Interessanterweise scheint die Menge und/oder Aktivität von BACE,
eines Enzyms welches entscheidend an der Aβ Herstellung beteiligt ist, in SMA Gehirnen
erhöht zu sein. Bisherige Untersuchungen nutzten transgene Mausmodelle oder Zelllinien,
jedoch ist die Übertragbarkeit der Ergebnisse vom Tier auf den Menschen fraglich, und
Zelllinien besitzen nur bedingt neuronale Eigenschaften. Als neuen Ansatz im Rahmen
dieser Doktorarbeit verwendeten wir deshalb die LUHMES Zelllinie, welche in vitro in
humane Neuronen ausdifferenziert werden kann. Zunächst etablierten wir ein neues,
2-stufiges Differenzierungsprotokoll. Wir konnten zeigen, dass sich die Vorläuferzellen
innerhalb von 5 Tagen irreversibel in postmitotische Neuronen umwandelten, begleitet von
ausgedehntem Neuritenwachstum und der Hochregulierung synaptischer Proteine. Die
neuronale Differenzierung wurde sowohl mit als auch ohne die Mediumsfaktoren cAMP und
GDNF ausgelöst, aber die zelluläre Produktion von Dopamin erforderte die Präsenz von
cAMP. Dabei beschrieben wir erstmals “nicht-dopaminerge“ LUHMES Neuronen, deren
Einsatz für verschiedenste Forschungsgebiete von Vorteil ist. Im nächsten Schritt
charakterisierten wird LUHMES als humanes Modell für MA-relevante Studien. Wir
untersuchten die Expression und Interaktion von Schlüsselproteinen wie Amyloid Precursor
Protein (APP) und BACE. Während der Langzeit-Zellkultur (10 Tage) stellten wir fest, dass
sowohl die Aβ Produktion als auch die Phosphorylierung von Tau beständig zunahmen, und
dass beide mit pharmakologischen Substanzen wie in Primärneuronen moduliert werden
konnten. Weiterhin fanden wir heraus, dass der Aβ Zunahme eine Aktivierung des RET
Rezeptors und damit des PI3K Signalwegs durch Wachstumsfaktoren wie GDNF zugrunde
lag. Das bedeutet, dass durch einen Anstieg von Wachstumsfaktoren, z.B. als
Schutzmechanismus im MA Gehirn, die Aβ Produktion weiter erhöht werden kann.
Schließlich etablierten wir eine stabile LUHMES Zelllinie mit BACE-Überexpression, um
seine erhöhte Aktivität bei SMA zu modellieren. Während der Differenzierung stieg der BACE
Level stetig an, was in einer entsprechend verstärkten β-Prozessierung von APP, und
zunächst auch in erhöhter Aβ Produktion resultierte. Nach Überschreiten eines bestimmten
BACE-Grenzwertes jedoch stellten die Zellen erheblich weniger Aβ als Wildtyp LUHMES her.
In diesen Zellen führte eine Abschwächung der BACE Aktivität, etwa durch Behandlung mit
niedrig dosierten BACE Inhibitoren, zu einem Anstieg von Aβ. Diese Ergebnisse steuern zum
Verständnis der komplexen Biologie der APP Prozessierung bei und könnten Auswirkungen
auf die Entwicklung von partiellen BACE Inhibitoren als MA Therapeutika haben.
2