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

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

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-173910

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

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

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

4.4 BACE ...10

4.5

5 Therapeutic aspects ...13

5.1 BACE inhibitors...13

5.2

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

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

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

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.

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.

B GENERAL INTRODUCTION

1 From normal aging to Alzheimer’s disease (AD)

The dramatic rise in life expectancy during the 20th century, from roughly 49 years to e.g. 80 years in Germany (CIA The World Factbook 2011), has resulted in a growing number of individuals achieving the age at which dementing disorders become common (Selkoe, 2001).

Among these, AD is the most frequent cause of dementia, estimated to contribute to 60-70%

of cases (Barker et al., 2002). With a worldwide prevalence of AD of approx. 30 million and an almost 4-fold rise of numbers expected by 2050, AD is likely to become one of the most important global public health issues (Holtzman et al., 2011).

Normal brain aging is connected to numerous structural and functional alterations of large variability. It is characterized by loss of brain volume (14% atrophy), neurons (10%) and synapses (up to 20%) (Caserta et al., 2009, Riederer et al., 2011). Brain aging is also linked to cognitive decline, and a major challenge is how to distinguish between normal and pathological aging, as manifested in AD. Since AD and aging are epidemiologically intertwined, they may share mechanistic commonalities. Nowadays it becomes more and more evident that AD is clinically not an all-or-nothing entity, but rather a continuum. The relevance of this notion is emphasized by the evolving concept of mild cognitive impairment (MCI) as transitional phase between normal aging and dementia (Petersen et al., 1999). At present, criteria for the clinical diagnosis of AD require that a patient has dementia before such a diagnosis can be made (Blennow, 2011). However, recent advances in the identification of AD biomarkers and in diagnostic tools such as magnetic resonance imaging (MRI) and positron-emission tomography (PET) have already made it possible to detect aspects of AD pathology in cognitively normal individuals and in patients with MCI (Hampel et al., 2010). The key insight of these studies is that the pathological changes that underlie the brain degeneration and cognitive loss in AD begin at least 10 to 20 years before dementia onset (Holtzman et al., 2011). Therefore, only last year, new definitions for AD have been proposed, which describe it as disease that encompasses both predementia and dementia phases (Dubois et al., 2010). Such redefined criteria of AD will most surely have a high impact on the diagnostic and therapeutic approaches to the disease.

2 Clinical symptoms and different forms of AD

The time course of AD dementia averages 7 to 10 years, and inevitably, the illness culminates in death. It is characterized clinically by progressive cognitive decline, and impaired recent memory is usually an initial symptom. Other cognitive deficits include changes in attention and problem-solving abilities, impaired judgment, decision-making and

orientation. As dementia progresses, language dysfunction and personality changes frequently appear (Galimberti and Scarpini, 2011, Holtzman et al., 2011).

AD is usually divided into two forms: familial cases with Mendelian inheritance of predominantly early-onset (< 60 years, FAD), and so-called sporadic cases with less apparent or no familial aggregation and usually later onset age (> 60 years, SAD) (Bertram et al., 2010). The genetic causes of FAD include dominant mutations of the genes coding for amyloid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2) (Goate et al., 1991, Levy-Lahad et al., 1995, Schellenberg et al., 1992). Although FAD accounts for only 5-10% AD cases, its pathological similarity to SAD suggests a common underlying mechanism that is simply accelerated in the context of certain genetic polymorphisms (Huse and Doms, 2000). Identification of causing factors for SAD is much more complicated.

Identification of specific risk genes is problematic because the overall increase in risk conferred by a single gene is small. Additionally, not just individual genes but combinations of risk alleles need to be identified (Ballard et al., 2011). The most consistently associated risk gene is ApoE. This gene encodes a protein, apolipoprotein E, which was previously recognized to play a role in cholesterol transport. Of the three ApoE alleles distributed throughout the population, the presence of two copies of the ApoE4 allele has been correlated with a more than seven times increased risk of developing SAD (Corder et al., 1993). More recently, the development of genome-wide arrays that allow the simultaneous evaluation of millions of single-nucleotide polymorphisms (SNPs) in thousands of samples revealed a number of new genetic risk factors. These are associated with genes encoding clusterin (CLU), phosphatidylinositol-binding clathrin assembly protein (PICALM), complement receptor 1 (CR1) and bridging integrator protein 1 (BIN1) (Harold et al., 2009, Lambert et al., 2009, Seshadri et al., 2010). In addition to risk genes, environmental factors like exposure to lead or other toxicants (Wu et al., 2008, Zawia and Basha, 2005) likely make an important contribution in determining an individual’s SAD risk, although the precise nature and mechanisms underlying this nongenetic components remain largely elusive (Bertram et al., 2010). Finally, posttranscriptional mechanisms, influencing the level, stability or activity of factors associated with AD, might play an important role. For instance, the increased activation of a translation initiation factor of BACE (one of the key enzymes of AD, see below), was suggested to be responsible for elevated levels of BACE in SAD (O'Connor et al., 2008).

3 Histopathological features of AD: plaques and tangles

The key neuropathological markers of AD were described by Alois Alzheimer in 1907 and at about the same time by Oskar Fischer (Goedert, 2009). At the macroscopic level, there is atrophy of the brain and extensive neuronal loss. Microscopically, the hallmarks of the

disease are extracellular amyloid plaques and intracellular neurofibrillary tangles (Fig. 1).

More recently recognized histopathologic features include synaptic degeneration, aneuploidy and inflammation (Galimberti et al., 2008, Swerdlow, 2007). Inflammation is mediated by activated microglia and astrocytes, which are frequently found around amyloid plaques (Nagele et al., 2004). It is still debated whether the role of glial cells, especially microglia, is a beneficial or detrimental one in AD, because it was shown that they are able to remove Aβ by phagocytosis but also that they might contribute to the development of new plaques and neurodegeneration (Nagele et al., 2004). A number of additional pathogenic mechanisms, possibly overlapping with plaque and NFT formation, have been described, including oxidative damage (Reddy et al., 2009) and mitochondrial dysfunction (Santos et al., 2010).

Figure 1. Pathological hallmarks of AD. From Irvine et al., 2008.

The principal components of amyloid plaques are the amyloid β (Aβ) peptides, which were originally isolated from cerebral vascular tissue of AD brains (Glenner and Wong, 1984).

These are protein fragments of 38- to 43-amino acids, which are present in plaques in aggregated forms including fibrils and oligomers (Kayed et al., 2003, Koffie et al., 2009). Why Aβ is so prone to aggregation is unclear, but its sequence, concentration, and destabilizing conditions are thought to be important factors (Nerelius et al., 2010). Various modifications of Aβ, taking place in or outside the cells, can also enhance its aggregation propensity, examples being phosphorylation (Kumar et al., 2011) and formation of pyroglutamate or isoaspartate (Saido et al., 1995, Shimizu et al., 2005). Although it is known that Aβ exerts cytotoxic effects on differentiated and aging neurons (Geula et al., 1998, Loo et al., 1993), it was for a long time unclear which Aβ form was responsible for these effects. While Aβ sequestered in plaques was at first proposed to represent the critical toxic species, more recent studies assume that actually the soluble oligomers are the ones to drive the disease (Lesne and Kotilinek, 2005, Walsh et al., 2005). In any case, according to the amyloid cascade hypothesis, Aβ is the initiating factor of AD (Hardy and Higgins, 1992, Selkoe, 1991), and plaque-oriented research dominated the AD field in the last two decades. In

recent years, however, emerging therapeutic strategies focused more and more also on the second important hallmark of AD: the neurofibrillary tangles (NFTs).

Beginning in the 1980s, immunocytochemical and biochemical analyses of NFTs suggested that they were composed of the microtubule-associated protein tau (Brion et al., 1985, Grundke-Iqbal et al., 1986, Kosik et al., 1988). Tau is synthesized and produced in all neurons and is also present in glia. In adult brain, there exist six isoforms of tau due to alternative mRNA splicing, which is developmentally regulated (Johnson and Stoothoff, 2004). Its most extensively described activity is to bind to tubulin and stabilize microtubules, although evidence for multiple other functions of tau exist (Morris et al., 2011). Despite a certain degree of phosphorylation also under physiological conditions, tau becomes abnormally hyperphosphorylated in AD. This might be a result of increased activity of tau kinases, e.g. glycogen synthase kinase-3β (GSK3β), possibly in combination with the deactivation of phosphatases like protein phosphatase 2A (PP2A). Aberrantly phosphorylated tau proteins detach from microtubules, thereby disrupting microtubule-based processes. Moreover, they might gain toxic functions by oligomerizing into cytotoxic species, as in the case of Aβ (Ballatore et al., 2007). The ultimately formed NFT aggregates have a high β-sheet content that ultrastructurally appears as paired helical filaments (PHFs) (Kidd, 1963). There is a typical progression of tau pathology within specific brain networks during the course of AD, starting first in areas such as the brainstem and transentorhinal region and then spreading throughout the limbic system and other areas of the neocortex (Braak and Braak, 1995, 1997). Although the regions of tau and Aβ pathologies only partially overlap, and the cause-and-effect relationship between them is still not exactly clear, it becomes increasingly evident that they influence and amplify each other (Ittner and Götz, 2011). In addition, there is emerging evidence from model systems that aggregates of both Aβ and tau, once formed, may spread from cell to cell in a prion-like fashion (Clavaguera et al., 2009, Eisele et al., 2010, Frost and Diamond, 2010). However, the tau and Aβ pathologies can also occur independently of each other. Tangles composed of tau aggregates have been described in more than a dozen less common neurodegenerative diseases, in almost all of which one finds no amyloid plaques (Selkoe, 2001). There are also infrequent cases of AD itself where only a few NFTs are found in the neocortex despite abundant Aβ deposits (Terry et al., 1987). Interestingly, Aβ accumulations can also be seen in the brains of cognitively normal-aged humans in the virtual absence of tangles. This finding, together with evolving data obtained from the application of new diagnostic tools (molecular neuroimaging, biomarker measurement etc.) lead several researchers to propose a diagram depicting the hypothetical sequence of biomarkers from the non-demented to AD stage (Craig-Schapiro et al., 2009, Holtzman et al., 2011, Jack et al., 2010, Perrin et al., 2009). According to this time- course, one of the first events to happen in people who are going to develop dementia

because of AD is the initiation of Aβ aggregation in the brain (Fig. 2). While people are still cognitively normal, amyloid plaques continue to accumulate. At some later point but still in the phase of preclinical AD, the formation of tau tangles starts to increase and neuronal integrity progressively deteriorates (Holtzman et al., 2011). By the time the earliest signs of cognitive decline are detectable, amyloid plaque deposition in the brain is already approaching its maximal extent whereas significant accumulation of NFTs does not peak until the stages of moderate to severe dementia (Fig. 2, lower dashed line) (Jack et al., 2010). The described scheme was however challenged by results showing that tau pathology in the entorhinal-hippocampal region clearly precedes Aβ accumulation by decades (Fig. 2, upper dashed line) (Braak and Braak, 1997, Duyckaerts, 2011). Additionally, abnormally phosphorylated tau material has very recently been detected within the brainstem of children and young adults in the absence of Aβ deposits (Braak and Del Tredici, 2011). In summary, although the order of occurrence of tau and Aβ pathologies still awaits further clarification, the data support the concept of preclinical Alzheimer’s disease (as mentioned in chapter 1), a phase during which plaques and NFTs accumulate for many years before the synaptic and neuronal loss they accompany manifest as cognitive decline (Morris and Price, 2001).

Figure 2. Proposed changes in Aβ plaque and tau tangle biomarkers in relation to the time course of clinical stages. The diagram was drawn according to Craig-Schapiro et al., 2009 and modified according to Duyckaerts, 2011.

4 Aβ generation and its key players

Aβ production and secretion is to a certain degree a normal metabolic event found to occur both in tissue culture and in the CNS, with concentrations of 10 to 20 ng/ml in the cerebrospinal fluid (CSF) (Haass et al., 1992, Seubert et al., 1992). Although a clear physiological function of Aβ is not yet established, several lines of evidence hint at a possible role in the control of synaptic activity (Kamenetz et al., 2003). Interestingly, in primary neuron cultures, the inhibition of endogenous Aβ production by secretase inhibitors or

immunodepletion of Aβ caused cell death, which could be prevented by the addition of physiological amounts of Aβ (Pearson and Peers, 2006, Plant et al., 2003).

Aβ is derived from APP through proteolytic processing by two enzymes: The first cut by BACE (β-secretase) releases a soluble APP fragment (sAPPβ) and leaves behind a C- terminal fragment (CTF). It was shown that BACE is able to cleave at two different sites of APP (position 1 or 11 of the Aβ sequence), giving rise to CTFs with either 99 (C99, Fig. 3) or 89 residues (Vassar et al., 1999). Alternatively, in a pathway that precludes Aβ formation, processing of APP by α-secretase results in a different soluble APP variant (sAPPα) and a shorter C-terminal fragment, C83 (Fig. 3) (Esch et al., 1990). In the next step, γ-secretase processes the CTFs (Fig. 3, not shown for C83), leading in the amyloidogenic pathway to the generation of an intracellular peptide called AICD (APP intracellular C-terminal domain) and Aβ (Fig. 3). Besides the N-terminal variations due to β-cleavage, Aβ can also have different C-terminal endings depending on the γ-cleavage site (Aβ40 or Aβ42, Fig. 3). The most abundant Aβ species produced in the brain and found in the CSF is Aβ1–40, whereas levels of the more readily aggregating Aβ_1–42 generally make up only 5 to 10% (Holtzman et al., 2011).

Figure 3.Schematic representation of APP processing by proteases.

See text for explanations.

4.1 Clearance of Aβ

Multiple enzymes within the central nervous system are capable of degrading Aβ, most of which are produced by neurons or glia (Miners et al., 2008). Three intensively studied peptidases are neprilysin (NEP), insulin-degrading enzyme (IDE) and endothelin-converting enzyme (ECE). While NEP is localized to synaptic membranes and therefore involved in degradation of extracellular Aβ (Fukami et al., 2002), IDE is predominantly found in the cytosol (Wang et al., 2006). ECE is, depending on its isoform, detected both at the cell surface and in acidic intracellular compartments like the trans-Golgi network (TGN) (Schweizer et al., 1997). Reductions in Aβ-degrading enzymes have been associated with possession of the APOE4 allele (Miners et al., 2008). The other two APOE alleles, on the contrary, seem to have a protective role in that they bind to Aβ and may favor its removal

form the brain (Bell et al., 2007, Holtzman et al., 1999). However, the major clearance transport mechanism of free monomeric Aβ is transcytosis across the blood brain barrier, which is mediated mainly by the low-density lipoprotein receptor related protein-1 (LRP1) (Deane et al., 2009, Shibata et al., 2000).

There is also growing evidence that in addition to its breakdown by specific proteases, Aβ can be degraded by the proteasome. In a cell-free assay, the 20S proteasome was shown to degrade both Aβ40 and Aβ42, and in in vitro and in vivo experiments, the inhibition of the proteasome resulted in significant increases in neuronal Aβ (Lopez Salon et al., 2003, Tseng et al., 2008). Vice versa, it appears that especially oligomeric forms of Aβ and also phospho- tau are able to block the ubiquitin-dependent protein degradation by the proteasome, thereby contributing to AD pathogenesis (Gregori et al., 1995, Keck et al., 2003, Oh et al., 2005).

Finally, since the lysosomal proteases cathepsin D and B have been shown to degrade Aβ (Hamazaki, 1996, Mueller-Steiner et al., 2006), it appears that the lysosomal system contributes under physiological conditions to Aβ clearance, for example via autophagy (Jaeger and Wyss-Coray, 2009).

4.2 APP

APP is an ubiquitously expressed member of a family of conserved type 1 transmembrane proteins, including also the APP like proteins 1 (APLP1) and 2 (APLP2) (Wasco et al., 1992, 1993). APP can, depending on alternative splicing, exist as either 770, 751, or 695 amino acid isoform (Selkoe, 1994). APP751 and APP770 splice forms are widely expressed in various tissues throughout the body, whereas neurons preferably express the 695 residue isoform (Sisodia et al., 1993). APP is cotranslationally translocated into the endoplasmic reticulum (ER) via its signal peptide and then posttranslationally modified during the transit through the secretory pathway, for example by the addition of N- and O-linked sugars in the ER and Golgi apparatus. Sulfation and phosphorylation in the late Golgi compartment and at the cell surface further contribute to the structural complexity of APP (Walter et al., 1997, Weidemann et al., 1989). Especially, phosphorylation of threonine 668 of APP is a posttranslational modification found selectively within neuronal growth cones and neurites (Ando et al., 1999, Iijima et al., 2000). This is in line with studies reporting that APP can be axonally transported by the fast anterograde component (Koo et al., 1990). It is present in vesicles in axonal terminals, although not specifically in synaptic vesicles, and can from there be retrogradely transported back to the cell body (Yamazaki et al., 1995). Although it has been assumed that the axonal terminals might be a principal site for the generation of Aβ from APP, this has not been definitively determined (Selkoe, 2001). It rather seems that APP which recycles back from the cell surface by endocytosis is subjected to β-cleavage in endosomal compartments (see chapter 4.4). The half-life of APP is relatively brief (20-30

min) (Weidemann et al., 1989) and its physiological function remains unclear. Recent evidence suggests that it plays a role in axonal pruning and neuronal migration during nervous system development (Nikolaev et al., 2009, Young-Pearse et al., 2010).

Furthermore, it has been implicated in cell- cell and cell-surface adhesion (Breen et al., 1991, Schubert et al., 1989), the stimulation of neurite outgrowth (Qiu et al., 1995, Small et al., 1994) and the regulation of intracellular calcium levels (Mattson et al., 1993). However, there is no evidence that a fundamental cellular function of APP is lost in AD patients (Selkoe, 2001). Information regarding the putative functions of APP cleavage products is also scarce, but it appears that e.g. AICD acts as transcriptional regulator (Gao and Pimplikar, 2001) and that sAPPα represents an autocrine (Saitoh et al., 1989) and neuroprotective factor (Mattson et al., 1993).

4.3 α-secretase

The identity of α-secretase has until now not been unequivocally established. A group of membrane bound metalloproteinases such as tumor necrosis factor-α converting enzyme (TACE) and members of the ADAM (a disintegrin and metalloproteinase) family, in particular ADAM9 and ADAM10, appear to be at least partially responsible for α-secretase cleavage (Buxbaum et al., 1998, Koike et al., 1999, Lammich et al., 1999). ADAM proteases are type I membrane proteins of the metzincin family and require a zinc ion for proteolytic activity (Edwards et al., 2008). ADAM substrates besides APP include N-cadherin and members of the epidermal growth factor family (Le Gall et al., 2009, Reiss et al., 2005).

Processing of APP by α-secretase occurs at the plasma membrane (Sisodia, 1992), and under certain conditions also in the TGN (Skovronsky et al., 2000). It is postulated to be protective in the context of AD because on the one hand, the enzyme cleaves within the Aβ sequence, thereby preventing its production, and on the other hand, the amount of neuroprotective sAPPα is increased (De Strooper et al., 2010, Lichtenthaler, 2011). Several studies have indicated that increased α-cleavage of APP reduces the processing by β- secretase and vice versa, suggesting that the two proteases compete for APP as substrate (Skovronsky et al., 2000, Vassar et al., 1999). However, this was not reproduced in all studies. For example, in several cell lines (HEK293, CHO and SH-SY5Y), no competition between constitutive α- and β-secretase cleavage was observable (Kim et al., 2008, Kuhn et al., 2010).

4.4 BACE

BACE (beta-site APP-cleaving enzyme, also called Asp2 or memapsin 2) is a 501 amino- acid type 1 transmembrane protein. This novel type of aspartic protease was more or less at

the same time identified as β-secretase candidate by expression cloning (Vassar et al., 1999), direct purification from human brain (Sinha et al., 1999), and bioinformatics (Hussain et al., 1999, Yan et al., 1999). A homolog of BACE named BACE2 has been identified with 64% amino acid sequence similarity, but this enzyme is expressed only at low levels in brain neurons and does not have the same cleavage activity on APP as β-secretase (Vassar et al., 2009). Although the majority of body tissues exhibit β-secretase activity (Haass et al., 1992), highest activity levels were observed in neural cell culture (Seubert et al., 1993). Also on mRNA and protein level, BACE has been reported to be abundant in the brain (De Strooper and Annaert, 2000, Marcinkiewicz and Seidah, 2000). There, it is mostly neurons which express BACE and therefore generate Aβ, at least under normal conditions. During AD- triggered inflammation, it is possible that glia, and astrocytes in particular, may produce significant levels of BACE and Aβ (Cole and Vassar, 2007).

Like all aspartic proteases, BACE is initially synthesized as a zymogen, containing a short prodomain. During translation in the ER, BACE undergoes core glycosylation at 3 or 4 N- linked sites. In the Golgi apparatus and during subsequent export through the secretory pathway, it further matures by the modification of the oligosaccharide chains and the removal of its 24-amino acid propeptide (Capell et al., 2000, Haniu et al., 2000). It appears that either the serine protease furin or another member of the proprotein convertase family is responsible for this cleavage (Bennett et al., 2000). Mature BACE is targeted to the cell surface and is then readily endocytosed again and delivered to endosomes. From there, it recycles either back to the cell surface or to the TGN. This trafficking seems to be regulated by the phosphorylation of its Ser 498 residue together with the presence of a C-terminal dileucine motif (He et al., 2005, Huse et al., 2000, Walter et al., 2001). BACE is also S- palmitoylated on 4 Cys residues located at the junction of the transmembrane and cytosolic domains (Benjannet et al., 2001, Vetrivel et al., 2009). This modification is in part responsible for the preferred localization of BACE within cholesterol-rich lipid rafts, where it cleaves raft- localized APP (Ehehalt et al., 2003, Riddell et al., 2001). Intracellularly, β-cleavage of APP predominantly takes place in the endosomal system and the TGN, since BACE functions optimally at low pH (Vassar et al., 2009, Yan et al., 2001). The enzyme has an unusually long half-life (between 12-16 h), as evidenced by pulse-chase experiments (Haniu et al., 2000, Huse et al., 2000), and is known to be degraded by either the lysosomal (Koh et al., 2005), or the ubiquitin-proteasomal pathway (Qing et al., 2004), or by endoproteolysis (Huse et al., 2003).

A variety of molecules have been shown to interact with BACE and increase its enzymatic activity, like prostate apoptosis response-4 (PAR-4) protein (Xie and Guo, 2005). On the other hand, certain other molecules were reported to inhibit the BACE-APP interaction and to thus reduce β-cleavage, e.g. sorLA/LR11 (Spoelgen et al., 2006) and heparan sulfate

(Scholefield et al., 2003). Recent studies have determined that levels of BACE activity and/or protein are elevated in aged healthy as well as in AD-affected brains (Fukumoto et al., 2002, 2004, Yang et al., 2003). Moreover, the Aβ load and C99 levels correlated with increased β- secretase activity in SAD patients (Holsinger et al., 2002, Li et al., 2004). Others demonstrated that disruption of the energy metabolism in APP transgenic mice, as simulation of the impaired glucose utilization in AD brains (de Leon et al., 1983), caused an increase in cerebral BACE and Aβ levels (Velliquette et al., 2005). As is the case for SAD, the BACE rise was not due to enhanced BACE gene transcription, but rather mRNA translation or protein stabilization. Taken together, these data strongly suggest that BACE is crucially involved in AD pathogenesis.

Regarding possible physiological functions of BACE, less is known. Recently, a role for BACE in brain development has been proposed, whereby BACE is required for the myelination and correct bundling of axons, probably by processing of the important factor neuregulin 1 (NRG1) (Hu et al., 2006, Willem et al., 2006). Further substrates of BACE include APLP1 and 2 (Li and Sudhof, 2004), low-density lipoprotein receptor-related protein (LRP) (von Arnim et al., 2005), P-selectin glycoprotein ligand-1 (PSLG-1) (Lichtenthaler et al., 2003) and a subunit of a voltage gated sodium channel (Wong et al., 2005). The characterization of these substrates will be useful for the evaluation of potential mechanism- based toxicities arising from inhibition of BACE and for implications regarding a larger potential role of BACE in other diseases in addition to AD.

4.5 γ-secretase

The identity of γ-secretase remained unknown for a long time. Based on pharmacological, genetic and cell biology studies, it is now clear that γ-secretase is a multi-subunit aspartyl protease (De Strooper et al., 2010). The complex comprises at least 4 proteins: PSEN1 or PSEN2, anterior pharynx-defective 1 (APH-1), presenilin enhancer protein 2 (PEN2), and nicastrin (De Strooper et al., 1998, Edbauer et al., 2002, Lee et al., 2002, Steiner et al., 2002). Besides APP, γ-secretase directly processes a number of other substrates like Notch, which is involved in cell fate decisions during development (Holtzman et al., 2011).

Generally, γ-secretase cleaves the hydrophobic integral membrane domain of its substrates, resulting in the release of protein fragments at both sides of the membrane (Annaert and De Strooper, 2002). The most important members of the enzyme complex are the presenilins, since they appear to form the catalytic core (Struhl and Greenwald, 1999, Wolfe et al., 1999).

PSEN1 and its close homolog PSEN2 define their own class of ubiquitously expressed membrane proteins (Levy-Lahad et al., 1995, Sherrington et al., 1995). Regarding their structure, different studies support a topology of 6-8 membrane-spanning regions (Dewji and Singer, 1997, Lehmann et al., 1997, Li and Greenwald, 1998). PSEN1 and 2 were reported

to be binding partners of a diverse group of proteins involved in processes ranging from cell adhesion to G-protein mediated signaling (Georgakopoulos et al., 1999, Smine et al., 1998, Stabler et al., 1999). Their assembly into γ-secretase is initiated in the ER and appears to be completed mainly in the intermediate compartment/cis-Golgi apparatus (De Strooper et al., 2010). The subcellular localization of the mature complex is, however, insufficiently studied.

What is known is mostly based on work with PSEN1 or on indirect evidence by locating Aβ.

PSEN1 is, surprisingly, mainly found in the ER (Annaert et al., 1999, Walter et al., 1996), and to a smaller extent on the cell surface and in endosomes (Rechards et al., 2003), whereas according to Aβ most of the γ-secretase cleavage of APP occurs in late compartments (Koo and Squazzo, 1994, Perez et al., 1999). This apparent “spatial paradox“ remains to be solved.

5 Therapeutic aspects

Treating AD is the biggest unmet medical need in neurology; current drugs like acetylcholine esterase inhibitors improve symptoms, but do not have profound disease-modifying effects (Citron, 2004). One promising concept for preventing and treating AD is based upon stimulating the immune system to remove Aβ from the brain. This can be done either in a passive way with Aβ antibodies, or by active immunization with human Aβ. Both approaches have been proven to be effective in clearing Aβ plaques and ameliorating cognitive deficits in mice (McLaurin et al., 2002, Schenk et al., 1999). After refinement of immunization methods became necessary due to adverse effects in a subset of patients in an initial clinical trial (Orgogozo et al., 2003), recent tests with e.g. bapineuzumab (a humanized anti-Aβ antibody) provided first direct therapeutic evidence of amyloid burden reduction in AD patients (Rinne et al., 2010). Bapineuzumab is currently being evaluated in phase III trials.

Another widely pursued strategy for the treatment of AD is prevention of Aβ production. To this end, approaches like substrate-based design or high-throughput screenings have been applied in the search for candidate β- and γ-secretase inhibitors (Woo et al., 2011).

5.1 BACE inhibitors

BACE is an attractive biological target, because its inhibition is expected to have limited side effects, but a challenging pharmacological target, because it possesses a large substrate binding site. This makes it difficult to generate highly selective inhibitors which are sufficiently potent but can nevertheless cross the blood brain barrier and penetrate the plasma/endosomal membranes (De Strooper et al., 2010). Alternatively, the delivery of an inhibitor to the intracellular compartments could be achieved by targeting the small fraction of

BACE that is localized to the cell surface. By binding to cell surface BACE, the drug would be delivered together with BACE to endosomes by endocytosis (De Strooper et al., 2010, Rajendran et al., 2008). Based on this mechanism of action, several peptidomimetic inhibitors have been developed, one example being OM99-2 (Hong et al., 2000). This is a hydroxyethylene isostere-based transition-state analogue inhibitor with excellent potency in vitro, yet its bulky structure precluded its application in vivo (Luo and Yan, 2010). Later generations of BACE inhibitors therefore comprised smaller, non-peptidic compounds. The pharmaceutical company Merck designed a non-traditional, hydroxyethylamine isophtalamide inhibitor named Merck-3 (in this thesis referred to as IPAD), which had nanomolecular potency in enzyme and cell-based assays (Stachel et al., 2004). Although Merck-3 was also shown to lower Aβ levels in vivo after intracranial administration, it was predicted to be not sufficiently CNS penetrant to be further tested (Stachel et al., 2006).

Johnson & Johnson on the other hand developed the aminodihydroquinazoline-derived potent BACE inhibitor 3a (in this thesis referred to as AQD) with excellent brain permeability and oral availability (Baxter et al., 2007). Although this compound, like most others, has never entered a clinical trial, recent reports on newly-developed small-molecule BACE inhibitors are encouraging. The company CoMentis recently announced the completion of the first phase I clinical trial of a candidate drug called CTS-21166 (Luo and Yan, 2010), and Evotec AG identified a series of BACE1 inhibitors by a novel screening method in conjunction with X-ray crystallography (Godemann et al., 2009).

5.2. γ-secretase inhibitors

Unlike β-secretase, γ-secretase has proved to be a highly tractable target for AD drug treatment, at least with respect to the development of orally bioavailable, brain-penetrant γ- secretase inhibitors (GSIs) (De Strooper et al., 2010). However, target-based toxicities related to inhibition of Notch cleavage by γ-secretase represent a major concern. For this reason, more recently developed compounds include so called Notch-sparing GSIs and γ- secretase modulators (GSMs). Only in July 2011, the company Bristol-Myers Squibb announced positive results of a phase II study evaluating the safety and tolerability of a Notch-sparing GSI named BMS-708163 in patients with mild-to-moderate AD, which had been demonstrated already in earlier studies to be highly selective, potent and well tolerable (http://www.alzforum.org/drg/drc/detail.asp?id=124). Non-steroidal anti-inflammatory drugs (NSAIDs) belong to the category of GSMs, because besides their beneficial effect on activated microglia and astrocytes they seem to shift γ-cleavage to less toxic Aβ species without affecting Notch cleavage (Heneka et al., 2011, Weggen et al., 2001). R-flurbiprofen, the first GSM to be evaluated in humans, was shown to be safe in the long term but failed in phase III clinical trials possibly due to low potency and poor CNS penetration (De Strooper et

al., 2010, Green et al., 2009). The two substances used in this thesis belong to the group of non-selective GSIs. LY450139, developed by Eli Lilly was tested in phase II trial in 2005 and was reported to reduce Aβ levels in plasma but not in CSF at concentrations that did not produce significant side effects (Klafki et al., 2006, Siemers et al., 2005, 2006). DAPT (N-[N- (3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) has been shown to be a potent GSI in a cellular system and has also proven in vivo efficacy in a mouse model (Dovey et al., 2001).

6 Model systems for AD research

In AD research, animal models find a wide-spread application. They aim to replicate the symptoms (cognitive dysfunction etc.), lesions (plaques and tangles) or the cause of the disease (risk factors, genes etc). In earlier years, procedures like brain destruction, treatment with exogenous chemicals or injection of Aβ were employed to mimic AD, but none of those models appeared sufficiently practical to be of common use (Duyckaerts et al., 2008). The real breakthrough came from the transgene technology, and various different transgenic mouse lines have since then been generated. The ability to study similar pathological processes in living animals has provided numerous insights into disease mechanisms (Spires and Hyman, 2005). Interestingly, overexpression of mouse APP does not cause Aβ deposition in mice, thus making transfection of human APP necessary. Even then, APP usually has to be mutated (to increase the extent of cleavage) to obtain reliable and abundant plaque formation (Duyckaerts et al., 2008). To mimic as many aspects of AD as possible, doubly (e.g. APP+PSEN1 or APP+BACE) transgenic mouse models appear to be best suited (McGowan et al., 2006). Although these have been successfully used in many Aβ-related studies, they rather model aspects of the human phase of “preclinical” AD, where clinically detectable symptoms have not yet arisen (Holtzman et al., 2011). A particular challenge has been that mice overproducing only Aβ-related genes fail to reproduce neuronal loss or NFT formation, even if hyperphosphorylated tau was detected with immunohistochemical methods. Therefore, the link between the alteration of APP metabolism and tau accumulation that has been postulated by the amyloid cascade hypothesis could so far not be reproduced, and the reason for this is still unknown (German and Eisch, 2004).

As alternative to the complicated and animal-consuming in vivo models, in vitro systems have been set up to investigate the physiopathological mechanisms involved in AD. Primary cultures of hippocampal slices have been extensively used to study the impact of Aβ oligomers on cell integrity and electrophysiology (Chen et al., 2000, Malouf, 1992, Varghese et al., 2010). Besides these, primary neurons from cerebral rat cortices (Greenfield et al.,

1999), and murine or rat cerebellar granule cells (CGC) (Pieri et al., 2010, Volbracht et al., 2009), as well as neuronal cell lines like human NT2N teratocarcinoma neurons (Chyung et al., 1997, Wertkin et al., 1993), mouse N2a neuroblastoma (Greenfield et al., 1999, von Arnim et al., 2006) and human SH-SY5Y neuroblastoma cells (Petratos et al., 2008, Schmechel et al., 2004, Xie et al., 1998) were applied. Even non-neuronal lines such as human (HEK293) or monkey (COS7) kidney cells (Benjannet et al., 2001, Bennett et al., 2000, Hoe et al., 2008, Huse et al., 2002, Lorenzen et al., 2010), fibroblasts (Kern et al., 2006) and Chinese hamster ovary (CHO) cells (Koh et al., 2005) gave the basis for numerous AD-related findings. Keeping in mind that 1. the neurons most affected in AD are those in or close to the cortical areas (entorhinal cortex, neocortex, hippocampus) and certain subcortical projection neurons such as basal forebrain cholinergic neurons (Holtzman et al., 2011), 2. the transfer of results from rodents to humans is often difficult and 3. there is concern about some abnormal functions in cancer cells, all the named cell lines represent in one way or the other suboptimal AD models. On the other hand, each of them possesses certain advantages like uncomplicated cell culture, high transfection efficiency, fast production of large cell numbers or neuronal features. The fact that the fundamental scheme of non-amyloidogenic and amyloidogenic APP cleavage has been set up based on experiments with HEK293 cells (Esch et al., 1990, Haass et al., 1992), and the large number of valuable insights that was gained with the different cellular systems suggest that certain compromises regarding AD-relevance are feasible.

7 The neuronal model system used in this thesis: LUHMES

LUHMES (Lund human mesencephalic) cells are a subclone of the originally generated MESC2.10 cell line (Lotharius et al., 2002). This cell line was obtained by preparation of precursor cells from embryonic ventral mesencephalic tissue and immortalization of these cells with of a LINX v-myc retroviral vector system (Hoshimaru et al., 1996). In this system, a tetracycline-controlled transactivator (tTA) strongly activates transcription of v-myc from a minimal CMV promoter in the absence of tetracycline. This allows the cells to continuously proliferate in a medium containing bFGF (Fig. 4). For initiation of differentiation, the cells are incubated with medium containing non-toxic concentrations of tetracycline, dibutyryl cyclic adenosine 3’,5’-monophosphate (db-cAMP) and glial cell line-derived neurotrophic factor (GDNF). Since tetracycline abolishes the transcription activation by tTA , the production of v- myc is blocked and the cells readily start to transform into post-mitotic neurons (Fig. 4) (Lotharius et al., 2002). In the following sections, more detailed information will be given on mesencephalic cells and the most important medium constituents.

Figure 4. Biphasic state of LUHMES:

growth and differentiation. See text for description. Abbreviations: bFGF: basic fibroblast growth factor, LTR: long terminal repeat, tTA: tetracycline- controlled transactivator (yellow circles), ires: internal ribosomal entry site, neo:

neomycin resistance gene, CMV:

cytomegalovirus-virus promoter, TET:

tetracycline (purple triangles), db:

dibutyryl, GDNF: glial cell line-derived neurotrophic factor. Slightly modified from Lotharius et al., 2002.

7.1 The ventral mesencephalon

The mesencephalon, or midbrain, is considered as part of the brainstem and is associated with multiple brain functions, such as vision, motor control, alertness and reward (Yin et al., 2009). The ventral (= anterior) mesencephalon is organized into different neuronal populations, including dopaminergic neurons and neurons of the red nucleus (RN) (Blaess et al., 2011). Dopaminergic neurons are further divided into anatomically and functionally distinct subclasses (Bjorklund and Dunnett, 2007). The substantia nigra (SN), located in the lateral-ventral midbrain, projects to the striatum and is involved in the regulation of motor behaviors. The ventral tegmental area (VTA), located more medially, projects to corticolimbic targets and is important for motivational states (Blaess et al., 2011). The functional diversity of these different regions becomes apparent in disease states: in Parkinson’s disease, SN neurons, but not VTA neurons, degenerate, resulting in severe motor deficits. In contrast, abnormalities in the corticolimbic system have been implicated in addiction and schizophrenia (Dagher and Robbins, 2009, Smidt and Burbach, 2007).

Dopaminergic and RN neurons have been shown to arise from ventral mesencephalic precursors that express sonic hedgehog (Joksimovic et al., 2009, Ono et al., 2007). Besides this, only little is known about the factors and genes that control the establishment of the distinct neuronal (sub)classes in the developing human brain (Nelander et al., 2009). In the mouse, the process has been subdivided into 3 major phases. First, the induction of a progenitor field within the neuroepithelium that is competent to generate DA precursors takes

place at an early stage of neural development (approximately at embryonic day 8.5) (Yin et al., 2009). Neurogenin 2 and Mash1 are considered as two of the early fate determinants of DA neurons (Ang, 2006). Following a second phase of specification and early differentiation, DA neurons terminally differentiate and acquire their mature phenotype (outgrowth of axons etc.) at relatively late stages of neurodevelopment (Prakash and Wurst, 2006). During this third phase, Nurr1 and Pitx3 were reported to play important roles (Smidt and Burbach, 2007).

7.2 The growth factor for proliferating LUHMES: bFGF

FGFs are polypeptides which play essential roles in a multitude of biological processes during development and adult life. Deregulation of FGF signaling, on the other hand, has been associated with many developmental syndromes, and with human cancer (Wesche et al., 2011). The FGF family consists of 22 members of closely related peptides which contain 150-300 amino acids and have a conserved core with ~30-60% identity (Itoh and Ornitz, 2004). They signal through 4 homologous high-affinity receptors (FGFR1-4) that have an overall structure similar to most receptor tyrosine kinases (Johnson and Williams, 1993). The prototypic FGFs, FGF1 and FGF2 (also named basic FGF = bFGF), were originally isolated from the brain and pituitary as mitogens for cultured fibroblasts (Gospodarowicz, 1975, 1978). They are paracrine factors, which do not possess a signal sequence for secretion, but utilize a non-classical secretion pathway circumventing the ER (Nickel, 2010).

FGFs are crucial during development, where they have been shown to be key molecules in organogenesis. FGF signaling is for example implicated in the formation of the heart, the lungs, the limbs and, most importantly, the nervous system, where it is implicated in neural induction (Dorey and Amaya, 2010, Powers et al., 2000, Turner and Grose, 2010). In cell culture, FGFs stimulate cell proliferation, survival, migration and differentiation (Dailey et al., 2005, Xian et al., 2005). FGF2 in particular has been shown to support the undifferentiated self-renewal of human embryonic stem cells and is routinely used to cultivate such cells in the laboratory (Lanner and Rossant, 2010).

7.3 The growth factor for differentiating LUHMES: GDNF

GDNF is a distant member of the transforming growth factor β superfamily and was originally isolated from a rat glial cell line (Lin et al., 1993). It is expressed throughout the central nervous system during development and in a more region-restricted manner also in the adult brain (Schaar et al., 1993, Stromberg et al., 1993). GDNF is known to be a potent survival factor for midbrain dopamine neurons both in vivo and in vitro (Lin et al., 1993, Pascual et al., 2008). The major source of GDNF to the midbrain is the striatum, from where it is retrogradely transported to the SN and the VTA (Barroso-Chinea et al., 2005, Tomac et al.,

GFRα1

1995). In addition to its dopaminotrophic function, GDNF plays an essential role in the development and survival of motor and sensory neurons and the development of the kidney (Moore et al., 1996, Pichel et al., 1996).

GDNF signals through a receptor tyrosine kinase named RET (rearranged during transfection), which was first discovered as a proto-oncogene (Takahashi, 2001). However, RET can only be activated, if GDNF first binds to a coreceptor, GFRα1 (GDNF-family receptor-α1). GFRα1 and RET are expressed in several brain regions in the developing and adult brain, including the cerebellum, hypothalamus and hippocampus, with particular abundance in the SN and the VTA (Carnicella and Ron, 2009). In the classical model, a GDNF dimer first binds to either monomeric or dimeric GFRα1, and the GDNF-GFRα1 complex then interacts with two RET molecules, thereby inducing their homodimerization and tyrosine autophosphorylation (Fig. 5) (Airaksinen et al., 1999). This leads to the downstream activation of several signaling cascades, such as the mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3K) and phospholipase Cγ (PLCγ) pathways (Fig.

5) (Manie et al., 2001, Wells and Santoro, 2009).

Figure 5. GFRα1 and RET mediated GDNF signaling pathways. Slightly modified from Carnicella and Ron, 2009. Autophosphorylation of tyrosine residues is indicated by red circles.

See text for explanations.

7.4 Second messenger supply from outside: db-cAMP

Many hormones and growth factors activate transcription by raising the level of cAMP within cells and thereby regulate proliferation, differentiation, survival and plasticity of cells by triggering programs of gene expression (Maruoka et al., 2010). The second messenger cAMP activates protein kinase A (PKA), which phosphorylates and regulates a variety of cellular proteins. One example is the transcription factor CRE binding protein (CREB), which then binds to the cAMP-responsive element (CRE), a consensus sequence found in promoter regions of many target genes (Johannessen et al., 2004). Regarding the brain- specific functions of cAMP, it has been suggested that cAMP-dependent mechanisms contribute to the maturation and maintenance of several catecholaminergic systems, including sympathetic ganglionic neurons and noradrenergic cells in the brainstem (Rydel and Greene, 1988, Sklair-Tavron and Segal, 1993). Furthermore, it has been shown that the

application of db-cAMP (a cell permeant analogue of cAMP) alone is sufficient to promote development and long-term survival of mesencephalic neuronal cultures (Hartikka et al., 1992, Michel and Agid, 1996). Finally, several groups reported that the extracellular supply of db-cAMP significantly potentiates the survival-promoting effects of GDNF on dopaminergic neurons and that CREB plays a crucial role in neurotrophin signaling (Bonni et al., 1995, Engele and Franke, 1996).

8 Aims of this thesis

Until the time point when this study was started, LUHMES had been used primarily in research related to their dopaminergic phenotype, especially in the Parkinson’s disease (PD) field (Christophersen et al., 2007, Lotharius et al., 2002, 2005, Paul et al., 2007). Notably, in one study they had been applied as tauopathy model for drug testing (Selenica et al., 2007), indicating that they might be suitable for investigations in the direction of AD. However, a comprehensive characterization of their neuronal and AD-relevant properties was still missing. Therefore, the aims of this thesis were:

1.] basic characterization of LUHMES cells as neuronal model system with emphasis on marker expression, neurite outgrowth and functional maturation during differentiation, and on a possible phenotype modulation by the omission of db-cAMP and GDNF from the culture medium

2.] evaluation of LUHMES in the undifferentiated and differentiated state regarding the expression and localization of AD-relevant proteins (esp. APP, BACE and tau) and the generation of APP cleavage products such as Aβ and sAPPα/β

3.] pharmacological and biological manipulation of tau phosphorylation and Aβ generation by different approaches and investigation of the effects exerted by medium constituents, especially GDNF, on APP processing and Aβ generation

4.] establishment of a LUHMES cell line with stable overexpression of BACE and the study of the possible influences on APP processing as an attempt to model conditions found in SAD

Chapter C

Rapid, complete and large-scale generation of post-mitotic neurons from the human LUHMES

cell line

Diana Scholz

, Dominik Pöltl

, Andreas Genewsky

, Matthias Weng

, Tanja Waldmann

, Stefan Schildknecht

and Marcel Leist

1Doerenkamp-Zbinden Chair for in vitro Toxicology and Biomedicine, University of Konstanz, Konstanz, Germany

2Konstanz Research School Chemical Biology, University of Konstanz

*These authors contributed equally to this work

J Neurochem (2011) 119, 957–971 (doi: 10.1111/j.1471-4159.2011.07255.x)

1 Abstract

We characterized phenotype and function of a fetal human mesencephalic cell line (LUHMES) as neuronal model system. Neurodevelopmental profiling of the proliferating stage (d0) of these conditionally-immortalized cells revealed neuronal features, expressed simultaneously with some early neuroblast and stem cell markers. An optimized 2-step differentiation procedure, triggered by shut-down of the myc transgene, resulted in uniformly post-mitotic neurons within 5 days (d5). This was associated with downregulation of some precursor markers and further upregulation of neuronal genes. Neurite network formation involved the outgrowth of 1-2, often > 500 µm long projections. They showed dynamic growth cone behavior, as evidenced by time-lapse imaging of stably GFP-overexpressing cells.

Voltage-dependent sodium channels and spontaneous electrical activity of LUHMES continuously increased from d0 to d11, while levels of synaptic markers reached their maximum on d5. The developmental expression patterns of most genes and of the dopamine uptake- and release-machinery appeared to be intrinsically predetermined, as the differentiation proceeded similarly when external factors such as dibutyryl-cAMP (cAMP) and GDNF were omitted. Only tyrosine hydroxylase required the continuous presence of cAMP.

In conclusion, LUHMES are a robust neuronal model with adaptable phenotype and high value for neurodevelopmental studies, disease modeling and neuropharmacology.

2 Introduction

Homogeneous cultures of human post-mitotic neurons are of interest in multiple research areas ranging from developmental neurobiology to toxicology. The demand on new model systems with regard to homogeneity and steady availability has increased. Furthermore, feasibility of molecular biological manipulations and the applicability of such cells for large screens are desirable, as such features have proven useful in studies unraveling mechanisms of genetic neurodegenerative diseases (Greer et al., 2010, Ittner et al., 2010).

Transformed cell lines provide the advantage of an easy supply, a relatively homogeneous culture, and the generation of genetically-modified subclones. For instance PC12, generated from a rat adrenal medullary pheochromocytoma, have greatly contributed to research on mechanisms of neurodegenerative diseases (Greene and Tischler, 1976, Rabizadeh et al., 1993, Xia et al., 1995) and neurotoxicology (Breier et al., 2010, Das et al., 2004). Their strict neurotrophin-dependence (Greene and Tischler, 1976) has been beneficial for some research questions, but also puts limits on the generalized use of the cells. Human cell lines derived from embryonic teratocarcinomas (e.g. NT2, hNT) (Pleasure et al., 1992) can be directed towards a post-mitotic neuronal phenotype, but the need for a very time-consuming differentiation protocol has limited their wide-spread use. Instead, human neuroblastoma cell lines, such as SH-SY5Y have been commonly applied in systematic toxicological evaluation programs (Forsby et al., 2009), as well as in studies of basic neurobiology (Biedler et al., 1978). Furthermore, they were used to examine the mechanisms of neurodegeneration (Tofaris et al., 2001) and for high throughput screenings (Loh et al., 2008), although they are hard to differentiate to a genuine post-mitotic state. The field of stem cell research may become the most important source for various human cell types in the future. Neurons of different specificity may be derived from human embryonic stem cells (Reubinoff et al., 2001), from adult neural stem cells (Johansson et al., 1999) or from human induced pluripotent stem cells (Lee et al., 2010). However at present, handling of such cell cultures is still time-consuming, associated with high costs, and neither homogeneity nor synchronization of cells are always given.

For the rational design of human neuronal models that address the present gaps, a scientific paradox has to be overcome: proliferation is needed to create large numbers, but neurons are by definition in a stable post-mitotic state. A successful solution is the transformation of committed neural precursor cells with myc oncogenes to ensure immortalization and continuous proliferation. Inactivation of the oncogene by exposure to neurotrophic factors (Donato et al., 2007) or tetracycline-controlled gene expression then allows neuronal differentiation. The latter approach is based on the retroviral LINX-v-myc vector with regulated v-myc expression (Hoshimaru et al., 1996). Addition of low concentrations of tetracycline abolishes v-myc expression, which allows cells to exit the cell cycle and to

differentiate. This construct was used to generate MESC2.10 cells to be used for neuronal transplantation in Parkinson’s disease (Lotharius et al., 2002). The source material was derived from 8-week old human ventral mesencephalic tissue. Karyotyping of the cell line showed a normal set of chromosomes and female phenotype (Paul et al., 2007). However, these cells were reported to be unstable and heterogeneous with regards to tyrosine hydroxylase (TH) expression and they were not suitable for replacement of dopaminergic (DA) neurons upon transplantation (Fountaine et al., 2008, Paul et al., 2007). In 2005, the subclone LUHMES (Lund human mesencephalic) was created (Lotharius et al., 2005) and used to study dopamine related cell death mechanisms (Lotharius et al., 2005, Schildknecht et al., 2009). With respect to the parkinsonian toxin 1-methyl-4-phenylpyridinium (MPP⁺), LUHMES behaved similar to primary cells (Schildknecht et al., 2009), while MESC2.10 were 1000-fold less sensitive (Fountaine et al., 2008). The general neuronal characteristics and the differentiation status of LUHMES at different culture conditions still await a comprehensive characterization. We addressed here the expression of neuronal markers, neurite outgrowth and electrophysiological properties of the cells, and present an optimized differentiation protocol leading to cultures with greatly improved homogeneity. The study also addressed the question whether functional and signaling studies may be performed with these cells in a simplified culture medium without added differentiation factors. These experiments revealed a robust, endogenous program driving the regulation of most neuronal genes independent of added cAMP/GDNF. Tyrosine hydroxylase was the most prominent exception, and required external signals. Therefore, the kinetics and conditions for up- and downregulation of this enzyme were further characterized.