Degeneration mechanisms in human dopaminergic neurons
Dissertation
zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.)
vorgelegt von
Dominik Pöltl
an der
Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie
Tag der mündlichen Prüfung: 2. August 2012 1. Referent: Prof. Dr. Marcel Leist 2. Referent: Prof. Dr. Martin Scheffner
Dominik Pöltl Degeneration mechanisms in human dopaminergic neurons
Publications integrated in this thesis
Chapter 3
Schildknecht S, Pöltl D, Nagel DM, Matt F, Scholz D, Lotharius J, Schmieg N, Salvo-Vargas A and Leist M. Requirement of a dopaminergic neuronal phenotype for toxicity of low concentrations of 1-methyl-4-phenylpyridinium to human cells. Toxicol Appl Pharmacol 2009;241:23-35.
Chapter 4
Pöltl D*, Scholz D*, Genewsky A, Weng M, Waldmann T, Schildknecht S and Leist M.
Rapid, complete and large-scale generation of post-mitotic neurons from the human LUHMES cell line. J Neurochem 2011;119(5):957-971. (* equal contribution)
Chapter 5
Pöltl D*, Schildknecht S*, Karreman C and Leist M. Uncoupling of ATP-depletion and cell death in human dopaminergic neurons. Neurotoxicology. 2011 Dec 19. [Epub ahead of print].
(* equal contribution)
Publications not integrated in this thesis
Weng MK, Zimmer B, Pöltl D, Broeg MP, Ivanova V, Gaspar JA, Sachinidis A, Wüllner U, Waldmann T and Leist M. Extensive transcriptional regulation of chromatin modifiers during human neurodevelopment. PLoS One 2012;7(5):e36708. Epub 2012 May 9.
Oral presentations
“LUHMES cells – a human in vitro model to study Parkinson’s disease”. Konstanz Research School Chemical Biology (KoRSCB) Retreat 2010, Schloss Hornberg, winner of MCAT speaker prize.
"LUHMES – a widely applicable neuronal model system", May 2011, Molekular- Neurologische Abteilung in der Neurologischen Klinik. Friedrich-Alexander-Universität Erlangen-Nürnberg / Universitätsklinikum Erlangen.
Posters
Dominik Pöltl, Stefan Schildknecht and Marcel Leist. “LUHMES Cells as Human In Vitro Model to Study Mechanisms of Parkinsonian Neurodegeneration”. World Parkinson Congress 2010, Glasgow, Schottland.
Stefan Schildknecht, Nina Stiegler, Diana Scholz, Dominik Pöltl and Marcel Leist.
“Application of human dopaminergic neurons for toxicity testing”. Congress on alternatives to animal testing 2010, Linz, Österreich.
Dominik Pöltl, Stefan Schildknecht and Marcel Leist. „LUHMES Cells as Human In Vitro Model to Study Mechanisms of Parkinsonian Neurodegeneration“. BWTOX.NET - Toxikologie Netzwerk Baden-Württemberg Symposium 2010, Freiburg, Deutschland.
Dominik Pöltl, Stefan Schildknecht and Marcel Leist. "Mechanistic studies on parkinonian neurodegeneration in LUHMES cells – a human in vitro model". 10th International Congress on Alzheimer's & Parkinson's Diseases 2011, Barcelona, Spanien.
Dominik Pöltl, Stefan Schildknecht and Marcel Leist. “Cell-death pathways downstream of mitochondria in human dopaminergic neuron degeneration”. BWTOX.NET - Toxikologie Netzwerk Baden-Württemberg Symposium 2011, Konstanz, Deutschland.
Stefan Schildknecht, Regina Pape, Dominik Pöltl, Diana Scholz, Marcel Leist. “Co-culture of human dopaminergic neurons and glia cells as in vitro model for Parkinson´s Disease.”
BWTOX.NET - Toxikologie Netzwerk Baden-Württemberg Symposium 2011, Konstanz, Deutschland.
Dominik Pöltl, Stefan Schildknecht, Christiaan Karreman and Marcel Leist. “Uncoupling of ATP-depletion and cell death in human dopaminergic neurons”. XIX World Congress on Parkinson’s Disease and Related Disorders 2011, Shanghai, China.
Stefan Schildknecht, Diana Scholz, Dominik Pöltl and Marcel Leist. “A human neuronal cell line for the substitution of transgenic neurodegenerative models”. 8th World Congress on Alternatives and Animal Use in the Life Sciences 2011, Montréal, Kanada.
Diana Scholz, Dominik Pöltl, Stefan Schildknecht and Marcel Leist. “Generation of post- mitotic neurons from the human LUHMES cell line”. 8th World Congress on Alternatives and Animal Use in the Life Sciences 2011, Montréal, Kanada.
Stefan Schildknecht, Regina Pape, Dominik Pöltl, Diana Scholz, Marcel Leist. “Co-culture of human dopaminergic neurons and glia cells as in vitro model for Parkinson´s Disease”. SOT (Society of Toxicology) 2011 - Annual Meeting, Washington, D.C., USA.
1 SUMMARY ... 13
ZUSAMMENFASSUNG ... 15
2 GENERAL INTRODUCTION ... 17
2.1 Parkinson’s Disease ... 17
2.1.1 Clinical features ... 17
2.1.2 Pathological features ... 19
2.1.3 Etiology of PD ... 19
2.1.4 Diagnosis of PD... 20
2.1.5 Molecular pathogenesis of PD... 21
Pathogenic mutations ... 22
Mitochondrial dysfunction and oxidative stress ... 23
Altered protein metabolism, misfolding and aggregation ... 24
Kinase-signaling pathways ... 25
Inflammatory changes ... 25
2.1.6 Treatment of PD ... 25
2.2 Model systems used to study PD ... 27
2.2.1 Animal models ... 27
2.2.2 Cellular models ... 29
2.2.3 LUHMES model... 31
2.2.4 Toxin models ... 32
6-OHDA, rotenone and paraquat ... 32
MPTP/MPP+ ... 33
2.3 Aims of the thesis ... 36
3 REQUIREMENT OF A DOPAMINERGIC NEURONAL PHENOTYPE FOR TOXICITY OF LOW CONCENTRATIONS OF 1-METHYL-PHENYLPYRIDINIUM TO HUMAN CELLS ... 37
3.2 Abstract... 38
3.3 Introduction ... 38
3.4 Materials and Methods ... 40
3.5 Results ... 47
3.5.1 Characterization of the model system ... 47
3.5.2 Pronounced MPP+-toxicity at micromolar concentrations ... 48
3.5.3 Time course of metabolic changes during MPP+-toxicity ... 49
3.5.4 Specificity of MPP+-toxicity to differentiated neurons ... 51
3.5.5 Requirement of a functional DAT for manifestation of MPP+-toxicity ... 52
3.5.6 The role of cellular dopamine synthesis for MPP+-toxicity ... 53
3.5.7 A potential role for the interaction of iron and MPP+ in LUHMES toxicity ... 55
3.5.8 Block of MPP+-driven degeneration by the mixed lineage kinase inhibitor CEP1347 ... 55
3.5.9 Live-cell multiparametric viability assay for further examination of MPP+-toxicity to LUHMES cells 57 3.6 Discussion... 59
3.6.1 Role of DA phenotype ... 59
3.6.2 MPP+ as inhibitor of mitochondrial respiration ... 62
3.6.4 Neuron-specific endpoints of toxicity and single cell imaging ... 63
3.7 Outlook ... 64
3.8 Acknowledgments ... 64
4 RAPID, COMPLETE AND LARGE-SCALE GENERATION OF POST-MITOTIC NEURONS FROM THE HUMAN LUHMES CELL LINE ... 65
4.1 Abstract... 66
4.2 Introduction ... 66
4.3 Materials and Methods ... 68
4.4 Results ... 74
4.4.1 Conversion of undifferentiated LUHMES into post-mitotic neuronal cells ... 74
4.4.2 Electrophysiological properties of post-mitotic LUHMES ... 75
4.4.3 Differential changes in phenotypic markers of neuronal maturation ... 77
4.4.4 Neurodevelopmental aspects of neurite growth ... 79
4.4.5 Progress along the dopaminergic lineage during LUHMES maturation ... 81
4.4.6 Robust predetermination of the neuronal fate under altered differentiation conditions ... 83
4.5 Discussion... 86
4.6 Acknowledgments ... 89
4.7 Supplementary figures ... 90
5 UNCOUPLING OF ATP-DEPLETION AND CELL DEATH IN HUMAN DOPAMINERGIC NEURONS ... 97
5.1 Abstract... 98
5.2 Introduction ... 98
5.3 Materials and Methods ... 100
5.4 Results ... 105
5.4.1 Basic characterization of the MPP+ cell death model in LUHMES neurons ... 105
5.4.2 Inhibition of cell death signaling on mitochondrial level ... 106
5.4.3 Protection by various pharmacological agents in the LUHMES/MPP+ model ... 108
5.4.4 Characterization of drugs preventing cell death of LUHMES ... 110
5.4.5 Uncoupling of cell death and ATP-depletion by pharmacologically distinct mechanisms ... 112
5.4.6 Uncoupling of cell death and ATP-depletion triggered by rotenone ... 113
5.5 Discussion... 114
5.6 Acknowledgments ... 117
5.7 Conflict of interest statement ... 117
6 GENERAL DISCUSSION ...119
6.1 Need for in vitro models in toxicological and pharmacological research ... 119
6.2 Model characterization ... 121
6.2.1 LUHMES recapitulate important steps of neuronal development and maturation ... 121
6.2.2 LUHMES as model for PD-related neurodegeneration ... 122
6.3 Susceptibility of DA neurons ... 124
6.4 Mechanisms of DA neuron degeneration ... 127
6.4.1 Mechanisms of MPP+-toxicity and protective strategies in LUHMES ... 128
DA system ... 128
Oxidative stress ... 128
Kinase signaling ... 129
Caspases ... 131
Mitochondria ... 131
Control compounds ... 133
Relevance for PD research and future therapies ... 133
6.5 Outlook & future applications of the LUHMES model ... 135
7 BIBLIOGRAPHY ...137
8 APPENDIX ...157
8.1 Record of contribution ... 157
8.2 Abbreviations ... 158
DANKSAGUNG ...159
Parkinson’s disease (PD) is the most common neurodegenerative movement disorder. The extensive loss of dopaminergic neurons in the Substantia nigra pars compacta and the resulting lack of dopamine in the target regions lead to severe motor symptoms. The single most consistent risk factor is aging. With a steadily increasing age of the world population, the prevalence of the global burden of PD will further rise in the future. So far, only symptomatic treatments that reduce motor complications are available. Neuroprotective and –restorative therapies are still lacking. To make things worse, at the time point of diagnosis around 60% of nigral dopaminergic neurons are already lost. Thus, strategies to prevent or at least to slow disease progression remain the central aim of PD-related research. We think that a deeper understanding of molecular mechanisms of the selective neurodegeneration is needed. Also, new and optimized models for the study of protective strategies are essential for further achievements in PD research. Animal models are often used, but differences in signaling pathways to humans exist. Furthermore, PD is a disease affecting solely humans. In vitro models, based on human material, represent a relevant alternative to in vivo experiments.
In this thesis, the human LUHMES cell line was introduced as model system for studies on neurodevelopment and neurodegeneration. We characterized the cell line with a focus on the suitability for PD research. We optimized the differentiation protocol and demonstrated a fast, homogeneous and irreversible differentiation to post-mitotic dopaminergic neurons. A distinct sensitivity to the parkinsonian model toxin 1-methyl-4-phenylpyridinium (MPP+) was shown.
This allowed mechanistic studies on parkinsonian neurodegeneration processes and the investigation of possible intervention strategies. The LUHMES/MPP+ model reproduced many molecular pathways of the complex disease. A newly established differentiation protocol, allowing the generation of dopamine-free LUHMES cells, demonstrated that dopamine contributes to MPP+-toxicity. Furthermore, different strategies using diverse classes of pharmacological inhibitors were found to protect LUHMES from MPP+-induced neurodegeneration.
Major findings were that (i) the LUHMES cell line represents an advanced in vitro model for PD research, and that (ii) dopaminergic neurons can be protected from degeneration, although a strong MPP+-mediated energy depletion via inhibition of complex I of the respiratory chain occured. We showed that ATP-depletion and cell death are not inevitably coupled.
Transferred to a general view on PD, with mitochondrial dysfunctions as key contributor to
disease progression, novel strategies for the protection from degeneration of dopaminergic neurons might be developed. The primary effect of MPP+ (in the cell model) and of mitochondrial dysfunction (in the disease), does not necessarily have to be prevented to be able to protect cells from dying. This approach might, in combination with an earlier diagnosis of the disease, make an important contribution to the development of neuroprotective therapies for PD.
Die Parkinson-Krankheit ist die häufigste neurodegenerative Erkrankung mit motorischen Störungen. Ein exzessiver Verlust von dopaminergen Neuronen in der Substantia nigra pars compacta und der damit einhergehende Dopaminmangel in den Zielregionen verursachen schwere motorische Komplikationen. Wichtigster Risikofaktor ist das Altern, daher ist in der immer älter werdenden Weltbevölkerung eine weitere Zunahme der Krankheitshäufigkeit unumgänglich. Die Parkinson-Krankheit wird deswegen auch in Zukunft eine der großen gesundheitlichen Herausforderungen unserer Gesellschaft darstellen. Therapeutische Maßnahmen ermöglichen bislang nur eine sympomatische Linderung der motorischen Störungen. Trotz intensiver Forschung konnten noch keine neuroprotektiven und -restorativen Maßnahmen entwickelt werden. Erschwerend kommt hinzu, dass zum Zeitpunkt der Diagnose bereits 60% der dopaminergen Neuronen der Substantia nigra degeneriert sind. Die Möglichkeit diese Krankheit zu verhindern oder zumindest die fortschreitende Degeneration zu verlangsamen stellt daher das zentrale Ziel der Parkinson-Forschung dar. Unserer Meinung nach liegt der Schlüssel zum Erfolg zum einen in einem tieferen Verständnis der molekularen Mechanismen des selektiven Degenerationsprozesses und zum anderen in der Entwicklung besserer Modelle für die Erforschung neuartiger Interventionsstrategien. Tiermodelle kommen zwar häufig zum Einsatz, stellen aber nicht zuletzt wegen Unterschieden in Signalwegen und der Tatsache, dass Parkinson eine rein humane Erkrankung ist, möglicherweise eine gute Annäherung, aber nicht unbedingt ein perfektes Modell dar. In in vitro Modellen hingegen können auch humane Zellen zum Einsatz kommen. Diese erlangen daher eine immer wichtigere Rolle in vielfältigen Bereichen der Forschung.
In dieser Arbeit wurde die humane LUHMES Zelllinie grundlegend und mit Fokus auf Parkinson-Forschung charakterisiert, ein optimiertes Differenzierungsprotokoll erarbeitet und das System wurde eingesetzt, um mögliche protektive Strategien vor Toxin-induzierter Neurodegeneration zu entwickeln. Wir charakterisierten die LUHMES Zelllinie ausführlich als Parkinson Modell und zeigten, dass eine sehr schnelle, homogene und irreversible Differenzierung zu post-mitotischen dopaminergen Neuronen möglich ist. Eine ausgeprägte Sensitivität gegenüber 1-Methyl-4-Phenylpyridinium (MPP+), einem Modell-Toxin der Parkinson-Forschung, wurde nachgewiesen und ermöglichte mechanistische Studien der Neurodegeneration, sowie die Erarbeitung von Strategien vor dieser zu schützen. Viele Aspekte der komplexen Erkrankung und viele in Patienten beschriebene beteiligte Signalwege
ermöglichte die Generierung von Dopamin-freien LUHMES Zellen. Mit Hilfe dieser Neuronen konnten wir zeigen, dass Dopamin selbst zur Sensitivität gegenüber MPP+ beiträgt.
Zusätzlich zeigten wir verschiedene Strategien auf, die es erlauben vor MPP+-induziertem Zelltod zu schützen, und fanden eine Vielzahl an Substanzklassen mit neuroprotektiver Wirkung. Obwohl MPP+ durch die Inhibition von Komplex I der Atmungskette stets zu einer starken Energiedepletion führte, können die Zellen durch verschiedene Substanzen vollständig geschützt werden.
Die wichtigsten Erkenntnisse dieser Arbeit waren, dass (i) LUHMES Zellen ein ausgereiftes System für in vitro Studien in der Parkinson-Forschung darstellen, und dass (ii) ATP- Depletion, ausgelöst durch mitochondriale Störungen, und Zelltod nicht zwangsläufig gekoppelt sein müssen. Überträgt man diese Erkenntnisse auf das Gesamtbild der Parkinson- Krankheit, in dem mitochondriale Fehlfunktionen als ein wichtiger Ursprung der Erkrankung angesehen werden, so könnten sich neue Strategien zum Schutz vor Degeneration dopaminerger Neuronen ergeben. Der primäre Effekt von MPP+ (im Zellmodell) bzw. von mitochondrialen Fehlfunktionen (in der Krankheit) muss also nicht zwangsläufig verhindert werden, um die Zellen vor dem Absterben zu schützen. Dieser Ansatz könnte im Idealfall mit einer früheren Diagnose der Erkrankung kombiniert werden und neue Therapiemöglichkeiten eröffnen und so einen wichtigen Beitrag im Kampf gegen Parkinson leisten.
This thesis is placed in the field of Parkinson’s disease (PD) research and uses a novel cell line as model to study molecular mechanisms that lead to degeneration of dopaminergic (DA) neurons. The understanding of principles underlying PD and general knowledge about the disease is essential and forms the basis of all research in this field. Therefore, not only molecular mechanisms are discussed, but also an extensive description of the syndrome PD is given in the first part of this work.
2.1 Parkinson’s Disease
According to the 2005 World Health Organization (WHO) report, neurodegenerative disorders are on their way to get ahead of cancer as second most common cause of deaths – after heart diseases – in the western world until 2040. Neurological disorders account for 11.2% of the global burden of disease in the European Region. With 12% of deaths globally, they are an important cause of mortality. Among these, 85% are contributed by cerebrovascular disease, 6.28% by Alzheimer’s Disease (AD) and other dementias, whereas 1.55% are associated with Parkinson’s Disease. PD strongly compromises quality of life and accounts for a substantial part of the global burden of neurological illnesses (WHO 2006).
The single most consistent risk factor is aging and with the steadily increasing age of the world population, the prevalence of PD will further rise in the future. PD is the second most common neurodegenerative disorder after AD. As most common neurodegenerative movement disorder it affects 1% of the population over 65 years of age (WHO 2006). PD was first described by James Parkinson in 1817 in “An Essay on the Shaking Palsy”. The striking core clinical feature of resting tremor is reflected by the name “Shaking Palsy” (Parkinson 1817). Seventy years later Jean-Martin Charcot suggested eternizing this researcher by renaming the disease into “Parkinson’s disease”. Great efforts were made since then in the field of PD research. Figure 1 shows some of the most important milestones. A chronological placement of the cellular model used in this thesis to study PD-related degeneration processes is also included.
2.1.1 Clinical features
PD is characterized by an extensive loss of dopaminergic neurons in the Substantia nigra pars compacta (SNpc). As a consequence, DA is not released to its target regions and severe motor symptoms (called “parkinsonisms”) are the result. Core clinical features of PD are resting
tremor is the most common and easy recognizable symptom of PD. Bradykinesia describes a slowness of movement and akinesia the absence of normal unconscious movements, e.g. arm swing during walking. Rigidity refers to an increased resistance/stiffness to passive movement of a limb (Dauer and Przedborski 2003). Postural instability may lead to falls and is generally a manifestation of the late stages of PD (Jankovic 2008). Freezing is another common symptom of PD. It impedes the beginning of voluntary movements, such as walking, and is one of the most disabling symptoms (Giladi et al. 2001). PD is the most common cause of parkinsonism, but also several other potential causes for this syndrome exist (Fahn 2003;
Tuite and Krawczewski 2007).
Figure 1: A selection of important events in the field of Parkinson’s disease research.
While the main motor symptoms of PD are related to degeneration of DA neurons, also other neurotransmitter systems (e.g. cholinergic, serotonergic, adrenergic) are affected in PD and degenerate during disease progression (Schapira 2009). A number of non-motor symptoms (e.g. fatigue, smell loss, constipation, sleep disturbances and behavioral/emotional dysfunctions) are the consequence. Unfortunately, some non-motor symptoms can also be caused by drug treatment of the motor symptoms (Tolosa E. 2010). Appropriate and early treatment would help to improve quality of life of the patients and reduce the costs of care of PD in society (Chaudhuri and Odin 2010; Tolosa E. 2010). Several non-motor symptoms (smell loss, rapid eye movement behavior disorder and constipation) are known to predate the motor signs and might help to determine the start of PD in a very early stage.
Mortality in PD patients is higher than in the control population. Before levodopa was discovered and introduced as therapy (see chapter 2.1.6), the mortality ratio of PD patients vs.
age- and sex-matched general population was 3:1 (Hoehn and Yahr 1967). The introduction of levodopa and other clinical progress halved mortality to a still increased rate of 1.52 for PD patients compared to age-matched population (Herlofson et al. 2004).
2.1.2 Pathological features
Pronounced degeneration of dopaminergic neurons in the SNpc and reduced levels of dopamine (DA) in the striatum is one key pathological feature of PD and the main cause of the motor features. When symptoms of PD become obvious, around 80% of DA is already depleted and around 60% of SNpc DA neurons have been lost (Dauer and Przedborski 2003).
Eosinophilic intra-neuronal proteinaceous inclusions, so-called Lewy bodies (LBs), are another pathological hallmark (Forno 1990). LBs and Lewy neurites were first described in 1912 by the neurologist Friedrich Lewy (Spillantini et al. 1997; Spillantini et al. 1998). The major constituent misfolded α-synuclein (ASYN) is associated with other proteins like ubiquitin. Exact mechanisms of formation are still unknown and whether those inclusions are rather neurotoxic or neuroprotective is still under debate (Olanow et al. 2004). Being the major component of LBs is one reason for the special attention ASYN got in PD research.
Braak and his colleagues found a correlation between the amount of insoluble ASYN deposition, its location, and the stage of the disease (Braak et al. 2002; Braak et al. 2003). LBs are also found in Dementia with Lewy bodies (DLB), but do not represent the pathological hallmark of any other degenerative disease besides PD. Another pathological feature of PD, besides protein aggregation and loss of DA signaling, is an active immune response. T-cell infiltration has been found in the SNpc of PD patient brains, an area which is normally immune privileged. Activated microglia and astrocytes have been shown to accumulate in and around the SNpc (Phani et al. 2012). But still, the question whether inflammation is the consequence of neurodegeneration or rather its cause needs more attention (Bornebroek et al.
2007; Khandelwal et al. 2011).
2.1.3 Etiology of PD
Parkinson’s disease is usually defined as an idiopathic disease, indicating that there is no known or identifiable cause. Exceptions were found in the recent years, when researchers identified several genes directly related to cases of PD (Polymeropoulos et al. 1997; Davie 2008). Genetic forms of PD are called “familial PD” and for truly idiopathic forms the term
“sporadic PD” will be used in the following.
Age remains the most prominent risk factor for PD, but the view on the etiology has dramatically changed in the last years. First, PD was thought to be purely sporadic. Then, it was realized that both environmental and genetic factors influence the disease. Nowadays, genetic predisposition is seen to increasingly contribute to the development of PD (Schapira and Jenner 2011). In 1996, the first genetic mutation of a single gene, sufficient to cause the PD phenotype, was mapped and identified as SNCA (α-synuclein (ASYN) gene) (Polymeropoulos et al. 1996; Polymeropoulos et al. 1997). For the first time, it was proven that PD may be hereditary. Besides missense mutations of SNCA, also duplications and triplications cause PD. Since then, the discovery of numerous other genes linked to rare familial forms of PD confirmed the role of genetics (see page 22, Pathogenic mutations).
Monogenic mutations account for 30% of familial and 3-5% of sporadic cases of PD (Klein and Westenberger 2012). Overall, the majority of PD cases are sporadic and only around 10%
show a strict familial etiology (Thomas and Beal 2007). Gene analysis revealed a mitochondrial damage repair pathway as one important pathway of pathogenesis, certainly involved in nigral cell death. PINK1, Parkin and DJ-1 are key players in this pathway (Hardy et al. 2009; Hardy 2010).
The study of PD-related genes helped to understand basic principles of the molecular pathogenesis (see chapter 2.1.5) of the more common sporadic forms of PD. Nevertheless, the etiology of this disease is not yet fully understood, but recognized as being multi-factorial and a combination of genetic susceptibilities and environmental factors.
2.1.4 Diagnosis of PD
Despite many years of intense research on diagnostic factors, there is no single definite test for the diagnosis of PD. Also no reliable biomarkers have been found yet and the diagnosis is still based on clinical features. A combination of cardinal motor symptoms, associated and exclusionary symptoms, as well as the response to levodopa are used for the diagnosis (Jankovic 2008). On clinical grounds, PD can be distinguished from atypical and secondary causes of parkinsonism (Halliday et al. 2011), but a definite test for PD is still urgently needed. Clinicopathological studies, which used brain bank material from Canada and the UK, demonstrated that the diagnosis of PD was incorrect in around 25% of patients (Tolosa et al. 2006). By the time motoric disabilities become obvious and the diagnosis of PD is possible, around 60% of DA neurons in the SN are already lost. To switch from symptomatic treatments to early initiated disease modifying therapies, an adequate method for early, ideally pre-symptomatic diagnosis, has to be established (DeKosky and Marek 2003). Promising
strategies focus on the evaluation of e.g. serum autoantibodies as biomarkers for the diagnosis for PD (Han et al. 2012).
Lack of DA in patients can be assessed by DAT-SPECT, single-photon emission computed tomography (SPECT) of the dopamine transporter (DAT). This highly sensitive method detects presynaptic dopaminergic deficits and helps to improve the accuracy of PD diagnosis (Kagi et al. 2010). Progression of PD symptoms is described and rated by the Hoehn and Yahr scale (Hoehn and Yahr 1967; Goetz et al. 2004), whereas the Braak staging is used to classify the degree of pathology in PD, but can be only performed by autopsy of the brain (Braak et al.
2002).
2.1.5 Molecular pathogenesis of PD
The cause of PD has been in the focus of neuroscientific research in the last decades, but still there is no proven neuroprotective and neurorestorative therapy for PD. Understanding molecular pathways and the biochemical background of PD is the basis for the development of effective therapeutics for this so far incurable disease. Until today, three major fields of research improved our understanding of the contribution of both genetic and environmental factors and their interplay in the development of PD. A big success story was the discovery of the toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, see page 33), selectively killing DA neurons, and consequential experimental models (Langston et al. 1984). Second, studies with human postmortem brain material provided new information about the causes of PD (Jenner et al. 1992). And third, genetic analyses led to the discovery that mutations in SNCA cause PD and opened new insights in the genetic basis of this disease (Polymeropoulos et al. 1997). The latter field, with immensely growing possibilities in sequencing technology, was the driving force in the understanding of the etiology in the past years (Schapira and Jenner 2011). It helped to identify key signaling pathways in the disease, necessary to develop targeted drugs (Gupta et al. 2008). Extremely similar phenotypes of familial and sporadic PD raise hope to be able to reduce the disease to a restricted number of common molecular pathways involved in disease progression (Gandhi and Wood 2005). Many genetic factors have been suggested to cause PD, but since some mutations demonstrate low penetrance (i.e.
the proportion of individuals carrying the mutation and exhibiting clinical symptoms is low) and disease concordance in relatives is also low, there must be interactions of multiple factors.
The multiple-hit, or gene-environment theory, suggests a combination of toxic stress (originating e.g. from mitochondrial dysfunction or DA oxidation) and the inhibition of neuroprotective cellular responses (like loss of stress-induced autophagic degradation or loss
Key players in cellular dysfunction in PD are mitochondrial malfunction, oxidative stress, disturbed protein metabolism and aggregation, dysfunction of the ubiquitin-proteasome system and autophagy, kinase signaling pathways, and inflammatory changes (Figure 2) (Dauer and Przedborski 2003; Gandhi and Wood 2005; Schapira and Tolosa 2010; Schapira and Jenner 2011). The mentioned points will be discussed in detail in the following.
Figure 2: Key players in the pathogenesis of PD. Inspired by (Fahn 2003).
Pathogenic mutations
Today, around 28 chromosomal regions related to PD are identified. The putative link of 18 of these loci to PD is highlighted by the PARK nomenclature (PARK1 – PARK18). Six of those regions contain genes that cause monogenic PD when mutated (Klein and Westenberger 2012). Genes with an autosomal dominant mode of inheritance are SNCA (PARK1/4) and LRRK2 (PARK8), autosomal recessive are Parkin (PARK2), PINK1 (PARK6), DJ-1 (PARK7) and ATP13A2 (PARK9) (Hardy et al. 2009). All of these are to a certain degree linked to mitochondrial processes (Finsterer 2011).
SNCA was the first gene associated with PD. So far, three different missense mutations – A53T (Polymeropoulos et al. 1997), A30P (Krüger et al. 1998), E46K (Zarranz et al. 2004) – as well as duplications and triplications of the whole gene are known to cause the disease (Singleton et al. 2003; Ibanez et al. 2004). ASYN protein is the main constituent of Lewy bodies, while its exact physiological function still remains unknown. ASYN possibly contributes to mitochondrial dysfunction and impaired protein degradation, is target of oxidative modifications and appears as aggregate in PD. Thus, it is part of all edges of the proposed “Bermuda Triangle” of insults leading to neuronal death in PD (Malkus et al. 2009).
Parkin encodes an E3 ligase, ubiquitinating and targeting misfolded proteins to the ubiquitin-
proteasome system for degradation. Mutations in this gene are the most common cause of early-onset PD (< 40 years of age) (Klein and Westenberger 2012). Loss-of-function mutations of PTEN-induced putative kinase 1 (PINK1) cause defective Parkin recruitment and dysfunctional Parkin-induced mitophagy, which is necessary for the elimination of damaged mitochondria (Youle and Narendra 2011). The partial mitochondrial localization of DJ-1 suggests a, so far unproven, role in mitochondrial functions. Mutations of this putative cellular sensor of oxidative stress also result in PD. Mutations in LRRK2 are the main cause of late-onset PD (> 40 years of age), but pathogenic mechanisms are not yet understood.
ATP13A2 mutations cause an atypical form of PD with juvenile-onset (< 20 years of age).
ATP13A2 is a lysosomal P-type ATPase of unknown function, which might play a role in mitochondrial quality control and increased reactive oxygen species (ROS) production when lost (Gusdon et al. 2012).
Taken together, all these genes are related to mitochondrial processes and highlight a special role of mitochondria in Parkinson’s disease.
Mitochondrial dysfunction and oxidative stress
Besides mutations of mitochondrial genes, also toxin-induced mitochondrial insults were found as cause for the disease. The discovery of the toxin MPTP, with its inhibitory function on mitochondrial complex I (Langston et al. 1983), led to a strong interest in mitochondria and suggested mitochondrial contribution to PD. Since then, several toxins are used in experimental models of PD (MPTP, Rotenone, 6-OHDA, Paraquat). All induce mitochondrial dysfunction, DA cell death and subsequent PD symptoms. This further highlights the pivotal role of mitochondria in PD (Betarbet et al. 2000; Thiruchelvam et al. 2000; Mounsey and Teismann 2010). Post-mortem studies using human brain material revealed deficits in complex I subunits and reduced activity in PD brain (Keeney et al. 2006). Also higher amounts of mitochondrial DNA (mtDNA) mutations in SNpc neurons were found in PD patients than in age-matched controls (Bender et al. 2006). Dysfunctional and reduced complex I activity leads to increased formation of ROS. Furthermore, mtDNA mutations and oxidative stress contribute to aging, the greatest risk factor of PD (Lin and Beal 2006). Cybrid cell lines (see chapter 2.2.2), containing mitochondria from PD patients, also show reduced complex I activity with increased ROS production (Swerdlow et al. 1996), increased proton leak and a reduction in maximum respiratory capacity (Esteves et al. 2010). Experimental inhibition of complex I was found to decrease proteasomal activity, which in turn can increase the vulnerability of neurons to normally subtoxic levels of free radicals (Höglinger et al.
activation of the intrinsic (mitochondrial) apoptosis pathway by the pro-apoptotic protein Bax and renders cells more prone to degeneration (Perier et al. 2005). Impaired mitochondrial dynamics (shape, size, distribution, movement), communication with other organelles, as well as quality control and turnover by autophagy (mitophagy) are also discussed in the molecular pathogenesis of PD (Schapira and Gegg 2011; Schon and Przedborski 2011).
As mitochondrial impairment is involved in both sporadic and familial forms of PD, mitochondrial dysfunction is considered as one critical element of the complex and multifactorial disease.
Altered protein metabolism, misfolding and aggregation
Neuronal homeostasis is disturbed as soon as protective mechanisms like protein degradation are not working correctly, are overloaded or unable to remove oxidized substrates. As a consequence, proteins might accumulate (Malkus et al. 2009). There are two major cellular pathways for protein removal and degradation: (i) the ubiquitin-proteasome system (UPS) and (ii) the autophagic-lysosomal system. Short-lived proteins and those misfolded in the endoplasmatic reticulum (ER) are removed by the UPS. Macro-, micro- and chaperone- mediated autophagy (CMA) demonstrate three types of autophagy, in which proteins and organelles are degraded in lysosomes. Dysfunctional UPS, macroautophagy, and CMA all demonstrate potential contributors to neurodegenerative diseases (Massey et al. 2006; Rogers et al. 2010). In PD, protein misfolding and aggregation play important roles. Three findings highlight a link between the ubiquitin-proteasome system and neurodegeneration in PD. First, Lewy bodies contain aggregated ASYN, ubiquitin – the signal for proteolysis by the proteasome –, and also proteasomes and heat-shock proteins. Second, two of the genes mutated in some forms of PD are enzymes of the ubiquitin-proteasome pathway (Parkin, UCH-L1). And third, post-mortem SN tissue analysis shows a significant reduction of proteasome activity in PD (Ross and Pickart 2004). Furthermore, age-related impairments of both the proteasomal and the lysosomal system are found in the aging brain (Keller et al.
2004). Disturbed ASYN degradation and subsequent accumulation has been implicated in PD pathogenesis. Under normal circumstances, ASYN is degraded by both proteasomal and autophagic-lysosomal pathways (Webb et al. 2003; Engelender 2012). Posttranslational modifications of ASYN can reduce its degradation, while dopamine-modification of ASYN blocks CMA of ASYN itself and of other substrates. This contributes to degeneration of DA neurons and may be one potential explanation for the selective vulnerability of DA neurons in PD (Martinez-Vicente et al. 2008).
Kinase-signaling pathways
Mitogen activated protein kinase (MAPK) pathways can be activated by a broad variety of stimuli and are involved in diverse cellular processes, as proliferation, differentiation, cell survival/demise, and apoptosis (Qi and Elion 2005). MAPKs are ubiquitously expressed in all tissue and are considered to be involved in most signal transduction pathways (Cuadrado and Nebreda 2010). MAPK pathways contain a tiered cascade of three kinases, sequentially activating each other by phosphorylation. A MAPK kinase kinase (MAPKKK) activates the MAPK kinase (MAPKK), which then activates the MAPK. The MAPK subsequently phosphorylates respective targets, including transcription factors, other kinases and enzymes (Bardwell 2006). In mammalian cells, five families of MAPKs have been identified: ERK1/2, JNK1/2/3, p38 kinase isozymes, ERK3/4 and ERK5. The first three are implicated in human diseases (Qi and Elion 2005). Members of the JNK family are of special interest in stress response in PD. Mixed-lineage kinases (MLKs) act as MAPKKK and activate MAPKK before JNK (a MAPK) is activated. Activation of JNK leads to phosphorylation of c-Jun, a transcription factor and important regulator of expression of apoptotic proteins (Wang et al.
2004). This pathway plays an essential role in many different models of parkinsonian neurodegeneration and is considered as important target for drug development (Silva et al.
2005).
Inflammatory changes
Also neuroinflammation gained a lot of attention in the last years in PD research. Activated microglia, T lymphocytes, as well as increased levels of pro-inflammatory cytokines are detected in the SN of PD patients (Hirsch et al. 2012). Post-mortem studies confirmed that both innate and adaptive immune response are involved. Humans exposed to MPTP showed activated microglia even many years after toxin exposure, indicating an ongoing inflammatory response (Langston et al. 1999; Hirsch et al. 2012). Despite being not considered as the one primary cause of PD, it is now clear that neuroinflammation is involved in the progression of neurodegeneration.
2.1.6 Treatment of PD
Currently, treatment options are limited. PD treatment is perforce symptomatic, since there is no proven neuroprotective or -restorative therapy. The developement of drugs that halt or retard disease progression is a major goal in PD research. Besides studies of genetic causes of PD, also post-mortem brain studies of sporadic PD helped to identify mitochondrial dysfunction, oxidative stress, altered protein metabolism and inflammation as key players in
the pathogenesis (Schapira and Tolosa 2010). Within these, converging pathways and interconnections at strategic points exist (Schapira 2010). A better understanding of PD pathogenesis is the basis for the development of new drugs, falling in one of the three categories of therapy: (i) symptomatic treatments, focusing on the alleviation of (mainly motor) symptoms, (ii) neuroprotective strategies, trying to prevent or reduce further cell loss, and (iii) neurorescue/-restorative approaches, aiming to restore normal function and enhance survival of dysfunctional neurons (Schapira 1999; Rodnitzky 2012).
(i) Symptomatic treatments include Levodopa (L-DOPA), the precursor of DA, which still is the most effective symptomatic oral medication for PD (Schapira et al. 2009). Since DA does not cross the blood brain barrier, it is given as precursor. Levodopa then is converted to DA mainly in DA neurons by the enzyme aromatic L-amino acid decarboxylase (AADC) (Schapira 2009). DA agonists (e.g. ropinirole, pramipexole) and monoamine oxidase B (MAO-B) inhibitors (e.g. selegiline, rasagiline) also demonstrate symptomatic relieve. DA agonists act by direct activation of DA receptors at the synapse and delay the onset of motor complications. MAO-B inhibitors reduce DA metabolism at the synapse and elevate its activity and re-uptake into DA neurons (Schapira 2009). New approaches of symptomatic treatments include adenosine 2a (A2a) antagonists, glutamate antagonists and serotonin receptor antagonists. In addition, new formulations of levodopa, with e.g. improved drug absorption or controlled release are tested (Rodnitzky 2012). Deep brain stimulation (DBS) via implantation of a pulse generator is another form of symptomatic treatment. It improves motor symptoms and quality of life, but is not curative. Ablative procedures, like pallidotomy and thalamotomy were widely replaced by DBS, as it is reversible and adjustable, and tries to modulate the function of a brain area, instead of destroying it (Machado et al. 2012). Since symptomatic treatments are not able to stop DA neuron degeneration, the long-term response is not satisfying.
(ii) Promising agents in neuroprotection are e.g. creatine, which is in is in phase III trials at the moment. Creatine inhibits the activation of the mitochondrial permeability transition pore and represses iron accumulation. Also minocycline, another phase III trial candidate, has neuroprotective abilities, probably mediated by its anti-oxidative, anti-inflammatory and iron chelating abilities. Furthermore, non-steroidal anti-inflammatory drugs (NSAIDS), nicotine and caffeine were found to lower the risk of PD in epidemiological studies (Seidl and Potashkin 2011).
(iii) Neurorestorative strategies include cell-based approaches and gene therapy. Cellular approaches try to simply replace degenerated neurons. Fetal DA neurons, transplanted in the striatum or in the striatum and the SN, already showed promising clinical results (Lindvall and Bjorklund 2004; Politis and Lindvall 2012). Nevertheless, tissue availability, ethical hurdles and the fact that transplanted fetal cells cannot be standardized, increase the need of alternatives. ESCs (embryonic stem cells), iPSCs (induced pluripotent stem cells), MSCs (mesenchymal stem cells) and NSCs (neural stem cells) might help to develop promising therapies in the future, but long-term safety and efficacy still have to be clarified (Pawitan 2011; Rascol et al. 2011). Gene therapy approaches are also in clinical trials and demonstrate the other arm of neurorestorative research. Under investigation are gene transfers of glutamic acid decarboxylase (GAD) using adeno associated virus (AAV), Neurturin (AAV-Neurturin = CERE-120), DOPA decarboxylase (AADC), or intrastriatal injection of a triscistronic lentivirus encoding TH, AADC and GPT cyclohydrase (Prosavin), leading to enhanced DA production at the injection site (Rodnitzky 2012). Some of these treatments have already shown promising results in human trials. RNA interference (RNAi) approaches are also established right now, to silence PD-associated genes like SNCA and LRRK2. But these are not yet in clinical trials.
Despite several promising approaches, there is still the unmet need of therapies that slow or stop progression of PD from an early stage on. Therefore, further knowledge about underlying molecular mechanisms and pathways is needed and can be gained using different models of parkinsonian neurodegeneration.
2.2 Model systems used to study PD
This chapter gives an overview of model systems used in PD research. Animal models are opposed to cellular models, the cell line used in this thesis is introduced and the most common toxin models of Parkinsonian neurodegeneration are explained.
2.2.1 Animal models
In PD research, there are essentially three kinds of experimental in vivo models. (i) Pharmacological, (ii) toxic and (iii) genetic models all help to understand the cause, mechanisms and treatment options of PD (Bezard and Przedborski 2011).
(i) Pharmacological models include e.g. reserpine- or alpha-methyl-tyrosin-treated rodents.
Reserpine depletes brain DA by inhibiting VMAT-2 (vesicular monoamine transporter 2),
screening of possible symptomatic efficacy of new PD drugs (Duty and Jenner 2011). The reserpine-treated rodent model is one of the oldest animal models in PD research and was fundamental in the development of L-DOPA (Carlsson et al. 1957), the standard treatment of motor symptoms. But since these models do not show degeneration of DA neurons, they lack the most prominent feature of PD.
(ii) A classical toxic PD animal model is based on nigral or striatal injection of 6-OHDA in rats. 6-OHDA leads to a selective destruction of the DA system, i.e. TH-containing neurons and their projections (Ungerstedt 1968). The rat model has been introduced in 1970 (Ungerstedt and Arbuthnott 1970) and later on, 6-OHDA has proven to be effective also in other animals (mice, cats, dogs, monkeys). Taken up via the dopamine transporter (DAT) into DA neurons, 6-OHDA induces degeneration mediated by oxidative stress and mitochondrial respiratory dysfunction. Subsequent degeneration of the nigrostriatal tract and reduced levels of striatal TH and DA lead to many features of PD, while LBs as pathological hallmark of PD are missing (Duty and Jenner 2011). The discovery of the shocking similarity of MPTP- intoxication with PD, led to the use of this compound in toxic models. Non-human primates treated with MPTP develop most clinical and pathological hallmarks of PD. Mice show a significant number of hallmarks, whereas rats are resistant to MPTP (Jackson-Lewis et al.
2012). MPTP is the gold standard animal model in PD. The lipopilic nature of MPTP allows systemic injection and rapid crossing of the blood brain barrier. MPTP is subsequently converted by astrocytic MAO-B to 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP+).
Spontaneous oxidation results in the active and toxic metabolite 1-methyl-4-phenylpyridinium (MPP+), which is selectively taken up into DA neurons via DAT. The MPTP primate model closely mimics PD features. Also all currently available PD treatments were shown to be successful in this system (Duty and Jenner 2011). But again, also in this model LBs are lacking. Other toxic models are based on the treatment with pesticides. Paraquat, a herbicide with structural similarity to MPTP/MPP+, generates massive oxidative stress by redox cycling and is often used in in vivo models. The highly lipophilic insecticide and herbicide rotenone readily crosses the blood brain barrier and seems to replicate important hallmarks of PD in rats, including highly selective DA neuron degeneration and LB-like cytoplasmic inclusions, containing ASYN and ubiquitin (Betarbet et al. 2000). More details about these toxins are found in chapter 2.2.4.
(iii) The trend in PD research goes to disease-gene based genetic animal models. Mainly mice are in use, but also non-mammalian models using Drosophila, C. elegans or zebrafish came
up. Transgenic mice models focus on SNCA knockout (KO) and expression of mutated forms, LRKK2 overexpression and KO, PINK1 KO and mutation, Parkin KO, and DJ-1 KO (Blandini and Armentero 2012). So far, the big break-through is still missing. At present, the best and most promising model might be the non-disease related genetic model of the MitoPARK mouse. In this model, mitochondrial function is selectively disrupted in DA neurons, allowing to focus on the respiratory chain defects observed in PD (Ekstrand et al.
2007).
No single model has shown all features of PD, but some are considered as adequate models and the closest we got to humans so far. As every system only represents some aspects of the very complex disease, the respective system has to be chosen according to the question of interest. Toxic models are useful when nigrostriatal lesions are required (but mostly lack LB pathology), while transgenic models are better suited for questions concerning selected molecular pathways (but mostly lack nigrostriatal DA neuron damage) (Blandini and Armentero 2012). Newer approaches try to increase the number of hallmarks of PD in the model by combining genetic mutations and neurotoxins in the same animal (Peng et al. 2010).
2.2.2 Cellular models
PD and most other human brain disease phenotypes are naturally not found in other species.
Therefore, the study of human patients, the use of human tissue, and cellular models with human origin are essential. Cellular models allow the study of specific pathways and single genes and proteins involved in the complex pathological processes of the multifactorial disease. PD-related alterations in biochemical processes are either induced by mutations of relevant genes or simulated by the use of PD-related toxins, like MPP+ or rotenone (Alberio et al. 2012). In vitro models comprise cell lines, primary cells and stem cells, while also mix- forms of these categories are evolving.
Neuroblastoma and carcinoma lines of human and non-human origin have a long tradition in neuroscience. PC12, a cell line derived from pheochromocytoma of the rat adrenal medulla, is commonly used (Greene and Tischler 1976). This is still one of the most used and cited cell systems in neuroscience, although being a tumor line, non-human and strictly dependent on nerve growth factor (NGF) for differentiation. Human neuroblastoma cell lines include e.g.
SHSY5Y, SK-N-BE, SK-N-MC, SK-N-SH and BE2-M17. Despite not being authentic DA neurons, SHSY5Y are commonly used to study PD-related questions. Due to their easy handling, they are suited for highly automated processes, like high-content screening systems
proliferative (tumor-like) or differentiated (neuron-like) cells for disease studies (Alberio and Fasano 2010).
Non-tumor cell lines can be generated from healthy fetal tissue by immortalization and differentiated to homogeneous populations of DA neurons. Examples are ReNcell VM NSCs (Donato et al. 2007), that derived from human fetal ventral mesencephalon and are immortalized via v-myc. MESC2.10 (Lotharius et al. 2002), as well as their subclone LUHMES (Lotharius et al. 2005), can be used as genetic and toxic model of PD and will be discussed in detail in chapter 2.2.3.
Patient-specific approaches use primary human skin fibroblasts as cellular model of PD.
Both aging of patients, plus genetic predisposition and environmental influences are represented by this model. Nevertheless, problems with clonal selection, drift in culture, extremely slow growth of primary cells from aged people, frequent mycoplasma contaminations, as well as broad differences in gene expression profiles and signaling pathways compared to neurons restricted their use (Auburger et al. 2012). Also whether fibroblasts are suited to mirror pathophysiological events of DA neurons of the human brain also needs to be further discussed. Patient-specific human cell lines can be based on reprogramming of human fibroblasts to iPS cells (induced pluripotent stem cells) or on so- called cybrids (hybrid cell lines). Cybrids are generated by fusing cells that lack mitochondrial DNA (mtDNA) with platelet mtDNA from PD patients (Schüle et al. 2009).
These lines reproduce phenotypic features of the disease and are e.g. used for studies of mitochondrial defects related to complex I activity (Ghosh et al. 1999). Those cells are suited to study mitochondrial deficits in PD and their role in disease progression, but remain technically challenging. In addition, the quality of cybrids depends on the quality and suitability of the recipient cell lines, which are often SHSY5Y or NT2 (Schüle et al. 2009).
Stem cell models represent another source of cells for PD research. Neural stem cells (NSCs) can be isolated from fetal and adult brain, expanded and further differentiated to neuronal and glial lineages and are of great interest for replacement therapies. To date, the probably most promising approach for studies concerning disease mechanisms and for drug screenings is the generation of disease-specific iPS cells (induced pluripotent stem cells) (Jang et al. 2012).
Adult human fibroblasts can be reprogrammed via overexpression of pluripotency-related transcription factors (OCT4, SOX2, KLF4, C-MYC) to establish hiPS cells (human iPS cells) (Takahashi et al. 2007). Different studies use hiPS from controls, sporadic and familial PD patients and the differentiation to DA neurons in order to find more pieces in the PD puzzle
(Devine et al. 2011; Nguyen et al. 2011; Seibler et al. 2011). Long-term cultures of hiPS from sporadic and familial PD patients developed signs of neurodegeneration (Sanchez-Danes et al.
2012). HiPS are useful for developing patient-specific drugs for personalized regenerative medicine, as they allow drug testing with a patient-specific genetic background.
Cellular models allow experiments in a highly controlled environment. Cell lines guarantee virtually unlimited material and the number of animals needed for experiments can be reduced. Furthermore, work with human material is more relevant in the development of drugs for human use. Of course, the whole complexity of PD cannot easily be studied in vitro.
But with the right choice of cellular model for the right question, the complexity of the disease might be broken down and underlying mechanisms might be solved step by step.
2.2.3 LUHMES model
All studies in this thesis were performed using LUHMES (Lund human mesencephalic) cells, a subclone of MESC2.10. MESC2.10 were created from 8-week old human ventral mesencephalic tissue (Lotharius et al. 2002) and showed a normal set of female chromosomes (Paul et al. 2007). The LUHMES subclone was created in 2005 (Lotharius et al. 2005) and shows morphological and biochemical features of mature dopaminergic neurons upon differentiation. MESC2.10, the progenitor line of LUHMES, was conditionally immortalized using the retroviral LINXv-myc vector (Figure 3A) for tetracycline-controlled v-myc expression (Lotharius et al. 2002). Briefly, this vector contains a tetracycline-controlled transactivator (tTA; originating from pUHD15-1 (Gossen and Bujard 1992)), that strongly activates the expression of v-myc (derived from pMC38 (Hoshimaru et al. 1996)). In the absence of tetracycline, expression is driven from a minimal promoter from human cytomegalovirus (hCMV), fused to the tetracycline operator sequence (Figure 3B). LUHMES can be kept in a proliferating state in medium supplemented with bFGF (basic fibroblast growth factor). Addition of low non-toxic concentrations of tetracycline shuts down v-myc expression and allows the cells to exit the cell cycle and to differentiate (Figure 3C).
Rapid division in the proliferation state allows to obtain large quantities of cells. Upon induction of differentiation, the cell cycle is left and an irreversible commitment to a stable post-mitotic neuronal phenotype takes place. These characteristics, required from an ideal neuronal cell line (Pleasure et al. 1992), are fulfilled by LUHMES. Furthermmore, LUHMES develop extensive neuritic processes and mature to a state equivalent to primary neuronal cultures.
Until the beginning of this thesis in 2009, LUHMES cells were not well characterized. Thus, one part of this study was a detailed and broad characterization of the LUHMES system.
Figure 3: Structure of LINXv-myc. (A) The long terminal repeat (LTR) transcribes the 7.8 kb mRNA. Assisted by the internal ribosome entry site (IRES), two proteins are produced: the tetracycline-controlled transactivator (tTA) and the neomycin phosphotransferase (neo). (B) tTA subsequently activates the tTA-dependent promoter (PhCMV*-1) and leads to the transcription of the 3.5 kb mRNA, resulting in the production of the v-myc protein. (C) Addition of tetracycline (TET) prevents tTA binding to the respective promoter and thereby prevents v-myc expression. Cells in status (B) proliferate, whereas cells in status (C) stop v-myc-driven proliferation and start to differentiate. Modified from (Hoshimaru et al. 1996).
2.2.4 Toxin models
PD models can be toxin-induced and genetic-based, or a combination of both. Genetic models are mainly based on the discovery of PARK genes and the (over)expression, knockout and knockdown of these or other putative PD genes. Many models are available both in vivo and in vitro. All experiments in this thesis were performed in a cellular model and are based on toxin-induced neurodegeneration. Thus, the focus in the following will be on toxin-based cellular systems. The most popular parkinsonian neurotoxins are 6-OHDA, rotenone, paraquat and MPTP (Figure 4) and will be discussed in the following sections.
6-OHDA, rotenone and paraquat
6-OHDA, as first DA neurotoxin discovered, is a prime example for an oxidative stress neurotoxin. It was introduced around 40 years ago and led to the destruction of adrenergic nerve endings (Thoenen and Tranzer 1968). Still actively used in PD research both in cellular and animal models, 6-OHDA does not cross the blood brain barrier (BBB). Due to the structural similarity to DA (see Figure 4), 6-OHDA enters DA neurons, where it generates ROS and quinones and produces superoxide and hydroxyl radicals (Bove et al. 2005; Miller et
al. 2009). 6-OHDA reproduces many symptoms resembling PD, but does not induce the formation of LBs.
The insecticide, pesticide and fish poison rotenone is a highly lipophilic compound which easily crosses the BBB and demonstrates a prime example of a mitochondrial toxin. It accumulates in mitochondria and inhibits complex I of the respiratory chain. Despite the uniform distribution throughout the brain, rotenone causes selective neurodegeneration of DA neurons (Miller et al. 2009).
Figure 4: Chemical structures of dopamine, 6-OHDA, paraquat, rotenone, MPTP, MPDP+ and MPP+.
Paraquat is a widely used herbicide with structural similarity to MPP+ (see Figure 4). It crosses the BBB via the neutral amino acid transporter and is selectively taken up into DA neurons via the dopamine transporter (DAT). Deleterious effects are mainly mediated by redox cycling with cellular diaphorase like NOS (nitric oxide synthase) (Bove et al. 2005), giving rise to paraquat radicals and eventually to hydroxyl free radicals and superoxide anions (Miller et al. 2009). Paraquat furthermore inhibits complex I of the mitochondrial respiratory chain and causes energy depletion as well as the generation of intracellular ROS (Miller et al.
2009).
MPTP/MPP+
In this study, mainly the neurotoxin MPP+ and its parental compound MPTP are used. Effects of MPTP on humans were discovered by chance in 1982, when several drug addicts in California developed severe parkinsonian symptoms. The contaminant MPTP, a by-product of chemical synthesis of MPPP – an analogue of meperidine (Demerol) – was identified as cause (Langston et al. 1983). Already back then, a selective damage of cells in the SN was proposed by Langston and his colleagues. MPTP-induced parkinsonism is almost indistinguishable from PD in humans and non-human primates, both by clinical and neuropathological aspects (Langston and Irwin 1986). Also responses to anti-parkinsonian therapies are almost identical to those observed in PD (Przedborski 2001). Experimental models showed that MPTP is
Przedborski 2003). Once inside the brain, MPTP is metabolized to the intermediate MPDP+ via MAO-B (Chiba et al. 1984; Heikkila et al. 1984) in non-DA cells, mainly astrocytes (Figure 5). Subsequently and most probably by spontaneous oxidation, MPP+ is formed and released or exported from those cells (Przedborski 2001). The parental compound MPTP is non-toxic to DA neurons, while MPP+ is actively transported in DA neurons via DAT (Mayer et al. 1986), where it unrolls its full deadly potential.
Figure 5: Schematic representation of MPTP metabolism. After systemic administration, MPTP crosses the blood brain barrier and is converted to MPDP+ by MAO-B in glial cells. The subsequent transformation into MPP+ and the release into the extracellular space are not yet fully understood. MPP+ is then actively taken up and concentrated into DA neurons via DAT.
Once inside DA neurons, MPP+ interacts with cytosolic enzymes, accumulates in mitochondria and is transported into DA storage vesicles via VMAT-2 (Przedborski 2001).
The redistribution of DA from vesicles to the cytosol leads to increased oxidative stress. The main toxic function of MPP+ is mediated by the inhibition of mitochondrial respiratory chain complex I (Nicklas et al. 1987) and leads to ATP-depletion and a massive generation of ROS.
Energy failure and oxidative stress are not killing directly, but rather trigger cell death-related molecular pathways and apoptotic programs, ultimately leading to cellular demise. A “circular cascade of deleterious events” starts (Przedborski et al. 2004), originating from and ending with mitochondria as central player. This process finishes with the activation of the programmed cell death machinery (Figure 6). Similar mechanisms are observed in post- mortem PD patient tissue, animal and cellular toxin models of PD. Following (programmed) cell death mechanisms are relevant in the MPTP/MPP+ model: MPP+-mediated inhibition of complex I, as well as redistribution of DA from storage vesicles to the cytosol both lead to an increase in ROS production (Vila and Przedborski 2003). Intramitochondrial oxidative stress
is increased and leads to an increase in the releasable soluble pool of cytochrome c within the mitochondrial intermembrane space (Perier et al. 2005). A subsequent damage of cellular components, including DNA, activates the tumor suppressor protein p53 (Mandir et al. 2002), the JNK/c-Jun pathway (Xia et al. 2001), and stimulates poly(ADP-ribose) polymerase (PARP) activity (Mandir et al. 1999). While p53 upregulates Bax, JNK contributes to the translocation of Bax to the mitochondrial membrane. Bax, in concert with activated PARP, induces the release of cytochrome c and apoptosis-inducing factor (AIF) to the cytosol (Vila and Przedborski 2003; Przedborski et al. 2004). Cytochrome c clusters with Apaf1 and pro- caspase 9 to form the apoptosome, initiating caspase 3-mediated cell death (Oettinghaus et al.
2012). In parallel, AIF contributes to caspase-independent mechanisms of cell death (Nicotra and Parvez 2002). Furthermore, reduced ATP levels, resulting from complex I inhibition, also contribute to cell death. Considering the toxic potential of MPP+, it is a fortunate circumstance that despite having been developed as a selective herbicide in the 1950ies (Cyberquat / Cyperquat, Gulf Oil Company), MPP+ never came on the market (Markey et al.
1984).
Figure 6: Schematic representation of mechanisms of MPP+ neurotoxicity within DA neurons. See text for details.
Following literature helped to design this overview: (Przedborski 2001; Dauer and Przedborski 2003; Vila and Przedborski 2003; Przedborski et al. 2004; Perier et al. 2007; Gorman 2008).
