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Sonstige beteiligte Institutionen
Summary
Bees, like many other organisms, evolved an endogenous circadian clock, which enables them to foresee daily environmental changes and exactly time foraging flights to periods of floral resource availability. The social lifestyle of a honey bee colony has been shown to influence circadian behavior in nurse bees, which do not exhibit rhythmic behavior when they are nursing. On the other hand, forager bees display strong circadian rhythms. Solitary bees, like the mason bee, do not nurse their offspring and do not live in hive communities, but face the same daily environmental changes as honey bees. Besides their lifestyle mason and honey bees differ in their development and life history, because mason bees overwinter after eclosion as adults in their cocoons until they emerge in spring. Honey bees do not undergo diapause and have a relatively short development of a few weeks until they emerge. In my thesis, I present a comparison of the circadian clock of social honey bees (Apis mellifera) and solitary mason bees (Osmia bicornis and Osmia cornuta) on the neuroanatomical level and behavioral output level.
I firstly characterized in detail the localization of the circadian clock in the bee brain via the expression pattern of two clock components, namely the clock protein PERIOD (PER) and the neuropeptide Pigment Dispersing Factor (PDF), in the brain of honey bee and mason bee. PER is localized in lateral neuron clusters (which we called lateral neurons 1 and 2: LN1 and LN2) and dorsal neuron clusters (we called dorsal lateral neurons and dorsal neurons: DLN, DN), many glia cells and photoreceptor cells. This expression pattern is similar to the one in other insect species and indicates a common ground plan of clock cells among insects. In the LN2 neuron cluster with cell bodies located in the lateral brain, PER is co-expressed with PDF. These cells build a complex arborization network throughout the brain and provide the perfect structure to convey time information to brain centers, where complex behavior, e.g. sun-compass orientation and time memory, is controlled. The PDF arborizations centralize in a dense network (we named it anterio-lobular PDF hub: ALO) which is located in front of the lobula. In other insects, this fiber center is associated with the medulla (accessory medulla: AME). Few PDF cells build the ALO already in very early larval development and the cell number and complexity of the network grows throughout honey bee development. Thereby, dorsal regions are innervated first by PDF fibers and, in late larval development, the fibers grow laterally to the optic lobe and central brain. The overall expression pattern of PER and PDF are similar in adult social and solitary bees, but I found a few differences in the PDF network density in the posterior protocerebrum and the lamina, which may be associated with evolution of sociality in bees.
Secondly, I monitored activity rhythms, for which I developed and established a device to monitor locomotor activity rhythms of individual honey bees with contact to a mini colony in the laboratory. This revealed new aspects of social synchronization and survival of young bees with indirect social contact to the mini colony (no trophalaxis was possible). For mason bees, I established a method to monitor emergence and locomotor activity rhythms and I could show that circadian emergence rhythms are entrainable by daily temperature cycles. Furthermore, I present the first locomotor activity rhythms of solitary bees, which show strong circadian rhythms in their behavior right after emergence. Honey bees needed several days to develop circadian locomotor rhythms in my experiments. I hypothesized that honey bees do not emerge with a fully matured circadian system in the hive, while solitary bees, without the protection of a colony, would need a fully matured circadian clock right away after emergence. Several indices in published work and preliminary studies support my hypothesis and future studies on PDF expression in different developmental stages in solitary bees may provide hard evidence.
Effects of dopamine on BDNF / TrkB mediated signaling and plasticity on cortico-striatal synapses
(2021)
Progressive loss of voluntary movement control is the central symptom of Parkinson's disease (PD). Even today, we are not yet able to cure PD. This is mainly due to a lack of understanding the mechanisms of movement control, network activity and plasticity in motor circuits, in particular between the cerebral cortex and the striatum. Brain-derived neurotrophic factor (BDNF) has emerged as one of the most important factors for the development and survival of neurons, as well as for synaptic plasticity. It is thus an important target for the development of new therapeutic strategies against neurodegenerative diseases. Together with its receptor, the Tropomyosin receptor kinase B (TrkB), it is critically involved in development and function of the striatum. Nevertheless, little is known about the localization of BDNF within presynaptic terminals in the striatum, as well as the types of neurons that produce BDNF in the cerebral cortex. Furthermore, the influence of midbrain derived dopamine on the control of BDNF / TrkB interaction in striatal medium spiny neurons (MSNs) remains elusive so far. Dopamine, however, appears to play an important role, as its absence leads to drastic changes in striatal synaptic plasticity. This suggests that dopamine could regulate synaptic activity in the striatum via modulation of BDNF / TrkB function. To answer these questions, we have developed a sensitive and reliable protocol for the immunohistochemical detection of endogenous BDNF. We find that the majority of striatal BDNF is provided by glutamatergic, cortex derived afferents and not dopaminergic inputs from the midbrain. In fact, we found BDNF in cell bodies of neurons in layers II-III and V of the primary and secondary motor cortex as well as layer V of the somatosensory cortex. These are the brain areas that send dense projections to the dorsolateral striatum for control of voluntary movement. Furthermore, we could show that these projection neurons significantly downregulate the expression of BDNF during the juvenile development of mice between 3 and 12 weeks.
In parallel, we found a modulatory effect of dopamine on the translocation of TrkB to the cell surface in postsynaptic striatal Medium Spiny Neurons (MSNs). In MSNs of the direct pathway (dMSNs), which express dopamine receptor 1 (DRD1), we observed the formation of TrkB aggregates in the 6-hydroxydopamine (6-OHDA) model of PD. This suggests that DRD1 activity controls TrkB surface expression in these neurons. In contrast, we found that DRD2 activation has opposite effects in MSNs of the indirect pathway (iMSNs). Activation of DRD2 promotes a rapid decrease in TrkB surface expression which was reversible and depended on cAMP. In parallel, stimulation of DRD2 led to induction of phospho-TrkB (pTrkB). This effect was significantly slower than the effect on TrkB surface expression and indicates that TrkB is transactivated by DRD2. Together, our data provide evidence that dopamine triggers dual modes of plasticity on striatal MSNs by acting on TrkB surface expression in DRD1 and DRD2 expressing MSNs. This surface expression of the receptor is crucial for the binding of BDNF, which is released from corticostriatal afferents. This leads to the induction of TrkB-mediated downstream signal transduction cascades and long-term potentiation (LTP). Therefore, the dopamine-mediated translocation of TrkB could be a mediator that modulates the balance between dopaminergic and glutamatergic signaling to allow synaptic plasticity in a spatiotemporal manner. This information and the fact that TrkB is segregated to persistent aggregates in PD could help to improve our understanding of voluntary movement control and to develop new therapeutic strategies beyond those focusing on dopaminergic supply.
Among mental disorders, panic disorder (PD) is one of the most common anxiety disorders characterized by recurring and unexpected episodes of extreme fear i.e. panic attacks. PD displays lifetime prevalence rates in the general population between 2.1-4.7 % and in about 30 to 40 % occurs comorbid with major depressive disorder (MDD). Differential methylation levels of the monoamine oxidase A (MAOA) gene have previously been associated with the etiology of both PD and MDD. The TGFB-Inducible Early Growth Response Protein 2 (TIEG2; alias KLF11), an activating transcription factor of the MAOA gene, has been reported to be increased in MDD, but has not yet been investigated in PD on any level.
Therefore, in an attempt to further define the role of an impaired TIEG2-MAOA pathway in anxiety and affective disorders, in the present thesis TIEG2 promoter DNA methylation was analyzed in two independent samples of I) PD patients with or without comorbid MDD in a case/control design and II) MDD patients with and without anxious depression. Additionally, in PD patients of sample I), TIEG2 methylation was correlated with Beck Depression Inventory (BDI-II) scores. Finally, in a third independent healthy control sample, correlation of TIEG2 promoter methylation levels with Anxiety Sensitivity Index (ASI) scores as a PD-related measure was analyzed.
No overall association of TIEG2 promoter methylation with PD was detected. However, PD patients with comorbid MDD showed significant TIEG2 hypomethylation compared to PD patients without comorbid MDD (p=.008) as well as to healthy controls (p=.010). In addition, MDD patients without anxious features displayed a statistical trend in decreased TIEG2 methylation in comparison to MDD patients with anxious depression (p=.052). Furthermore, TIEG2 methylation was negatively correlated with BDI-II scores in PD patients (p=.013) and positively correlated with ASI scores in the healthy control sample (p=.043).
In sum, the current study suggests TIEG2 promoter hypomethylation as a potential epigenetic marker of MDD comorbidity in PD or of non-anxious depression, respectively. If replicated and verified in future studies, altered TIEG2 methylation might therefore represent a differential pathomechanism of anxiety and mood disorders.
Background
- Brain-Computer Interfaces (BCI) enable their users to interact and communicate with the environment without requiring intact muscle control. To this end, brain activity is directly measured, digitized and interpreted by the computer. Thus, BCIs may be a valuable tool to assist severely or even completely paralysed patients. Many BCIs, however, rely on neurophysiological potentials evoked by visual stimulation, which can result in usability issues among patients with impaired vision or gaze control. Because of this, several non-visual BCI paradigms have been developed. Most notably, a recent study revealed promising results from a tactile BCI for wheelchair control. In this multi-session approach, healthy participants used the BCI to navigate a simulated wheelchair through a virtual apartment, which revealed not only that the BCI could be operated highly efficiently, but also that it could be trained over five sessions. The present thesis continues the research on this paradigm in order to - confirm its previously reported high performance levels and trainability - reveal the underlying factors responsible for observed performance increases - establish its feasibility among potential impaired end-users
Methods
- To approach these goals, three studies were conducted with both healthy participants and patients with amyotrophic lateral sclerosis (ALS). Brain activity during BCI operation was recorded via electroencephalography (EEG) and interpreted using a machine learning-based linear classifier. Wheelchair navigation was executed according to the classification results and visualized on a monitor. For offline statistical analysis, neurophysiological features were extracted from EEG data. Subjective data on usability were collected from all participants. Two specialized experiments were conducted to identify factors for training.
Results and Discussion
- Healthy participants: Results revealed positive effects of training on BCI performances and their underlying neurophysiological potentials. The paradigm was confirmed to be feasible and (for a non-visual BCI) highly efficient for most participants. However, some had to be excluded from analysis of the training effects because they could not achieve meaningful BCI control. Increased somatosensory sensitivity was identified as a possible mediator for training-related performance improvements. Participants with ALS: Out of seven patients with various stages of ALS, five could operate the BCI with accuracies significantly above chance level. Another ALS patient in a state of near-complete paralysis trained with the BCI for several months. Although no effects of training were observed, he was consistently able to operate the system above chance level. Subjective data regarding workload, satisfaction and other parameters were reported.
Significance
- The tactile BCI was evaluated on the example of wheelchair control. In the future, it could help impaired patients to regain some lost mobility and self-sufficiency. Further, it has the potential to be adapted to other purposes, including communication. Once visual BCIs and other assistive technologies fail for patients with (progressive) motor impairments, vision-independent paradigms such as the tactile BCI may be among the last remaining alternatives to interact with the environment. The present thesis has strongly confirmed the general feasibility of the tactile paradigm for healthy participants and provides first clues about the underlying factors of training. More importantly, the BCI was established among potential end-users with ALS, providing essential external validity.
Comparative analysis of insect circadian clocks: a behavioural, anatomical, and molecular study
(2020)
Biological clocks are endogenous oscillators that give organisms the sense of time. Insects, as the largest taxonomic group, offer fascinating models to study the evolution of clocks and their adaptation to various environments. Although the laboratory fruit fly, Drosophila melanogaster, led the role in the field of circadian biology as it provides a powerful genetic experimental tool, new model insect species need to be established to understand photoperiodic responses and to enable comparative studies. This work reports the behavioural, anatomical, and molecular characterization of the circadian clock of five insect species. The malt fly Chymomyza costata carries a D. melanogaster-like clock network, which supports circadian rhythms under rhythmic environment but cannot self-sustain when isolated from external time cues. The olive fly Bactrocera oleae is the major pest of olive plantations and the characterization of its circadian clock will improve future pest management strategies. The linden bug Pyrrhocoris apterus, a well suited model for investigating circadian and photoperiodic timing interactions, shows high degree of homology of the clock network with D. melanogaster. The scuttle flies Megaselia scalaris and Megaselia abdita represent new fascinating models to study how the clock network controls circadian behaviour. Overall, this work highlights high degree of homology between different circadian clock systems, but at the same time also dramatic differences in terms of circadian behaviour and neuro-anatomical expression of clock components. These have been mainly discussed in regards to the evolution of clocks in Diptera, and the adaptation of clocks to high latitudes.
We are living in a system that underlies permanent environmental changes due to the rotation of our planet. These changes are rhythmic with the most prominent one having a period of about 24 hours, but also shorter and longer rhythms characterize our environment. To cope with the ever-changing environmental conditions, it is thought to be beneficial if an organism can track and anticipate these changes. The so called endogenous clocks enable this and might provide a fitness advantage. To investigate and unravel the mechanism of endogenous clocks Chronobiologists have used different model organisms. In this thesis Drosophila melanogaster was used as model organism with its about 150 clock neurons representing the main endogenous clock of the fly in the central brain.
The molecular mechanisms and the interlocked feedback loops with the main circadian key players like period, timeless, clock or cycle are under investigation since the 1970s and are characterized quite well so far. But the impact of a functional endogenous clock in combination with diverse factors and the resulting fitness advantages were analysed in only a few studies and remains for the most part unknown. Therefore the aim of this thesis was to unravel the impact of Drosophila melanogaster`s endogenous clock on the fitness of the fly. To achieve this goal different factors – like day length, humidity and food composition – were analyzed in wild type CS and three different period mutants, namely perL, perS and per01, that carry a point mutation altering or abolishing the free-running period of the fruit fly as well as a second arrhythmic strain, clkAR.
In competition assay experiments wild type and clock mutant flies competed for up to 63 generations under a normal 24 hour rhythm with 12 hours light/day and 12 hours darkness/night (LD12:12) or T-cycles with 19 or 29 hours, according to the mutants free-running period, or constant light (LL) in case of the arrhythmic mutant as well as under natural-like outdoor conditions in two consecutive years. Overall the wild type CS strain was outcompeting the clock mutant strains independent of the environmental conditions. As the perL fly strain elongated their free-running period, the competition experiments were repeated with naturally cantonized new fly strains. With these experiments it could be shown that the genetic background of the fly strains – which are kept for decades in the lab, with backcrosses every few years – is very important and influences the fitness of flies. But also the day length impacts the fitness of the flies, enabling them to persist in higher percentage in a population under competition. Further factors that might influence the survival in a competing population were investigated, like e.g. mating preferences and locomotor activity of homo- and heterozygous females or sperm number of males transferred per mating. But these factors can still not explain the results in total and play no or only minor roles and show the complexity of the whole system with still unknown characteristics.
Furthermore populations of flies were recorded to see if the flies exhibit a common locomotor activity pattern or not and indeed a population activity pattern could be recorded for the first time and social contact as a Zeitgeber could be verified for Drosophila melanogaster.
In addition humidity and its impact on the flies´ fitness as well as a potential Zeitgeber was examined in this thesis. The flies experienced different relative humidities for eclosion and wing expansion and humidity cycle phase shifting experiments were performed to address these two different questions of fitness impact and potential Zeitgeber. The fruit fly usually ecloses in the morning hours when the relative humidity is quite high and the general assumption was that they do so to prevent desiccation. The results of this thesis were quite clear and demonstrate that the relative humidity has no great effect on the fitness of the flies according to successful eclosion or wing expansion and that temperature might be the more important factor. In the humidity cycle phase shifting experiments it could be revealed that relative humidity cannot act as a Zeitgeber for Drosophila melanogaster, but it influences and therefore masks the activity of flies by allowing or surpressing activity at specific relative humidity values.
As final experiments the lifespan of wild type and clock mutant flies was investigated under different day length and with different food qualities to unravel the impact of these factors on the fitness and therefore survival of the flies on the long run. As expected the flies with nutrient-poor minimum medium died earlier than on the nutrient-rich maximum medium, but a small effect of day length could also be seen with flies living slightly longer when they experience environmental day length conditions resembling their free-running period. The experiments also showed a fitness advantage of the wild type fly strain against the clock mutant strains for long term, but not short term (about the first 2-3 weeks).
As a conclusion it can be said that genetic variation is important to be able to adapt to changing environmental conditions and to optimize fitness and therefore survival. Having a functional endogenous clock with a free-running period of about 24 hours provides fitness advantages for the fruit fly, at least under competition. The whole system is very complex and many factors – known and unknown ones – play a role in this system by interacting on different levels, e.g. physiology, metabolism and/or behavior.
Endogenous clocks regulate physiological as well as behavioral rhythms within all organisms. They are well investigated in D. melanogaster on a molecular as well as anatomical level. The neuronal clock network within the brain represents the center for rhythmic activity control. One neuronal clock subgroup, the pigment dispersing factor (PDF) neurons, stands out for its importance in regulating rhythmic behavior. These neurons express the neuropeptide PDF (pigment dispersing factor). A small neuropil at the medulla’s edge, the accessory medulla (AME), is of special interest, as it has been determined as the main center for clock control. It is not only highly innervated by the PDF neurons but also by terminals of all other clock neuron subgroups. Furthermore, terminals of the photoreceptors provide light information to the AME. Many different types of neurons converge within the AME and afterward spread to their next target. Thereby the AME is supplied with information from a variety of brain regions. Among these neurons are the aminergic ones whose receptors’ are expressed in the PDF neurons. The present study sheds light onto putative synaptic partners and anatomical arrangements within the neuronal clock network, especially within the AME, as such knowledge is a prerequisite to understand circadian behavior. The aminergic neurons’ conspicuous vicinity to the PDF neurons suggests synaptic communication among them. Thus, based on former anatomical studies regarding this issue detailed light microscopic studies have been performed. Double immunolabellings, analyses of the spatial relation of pre- and postsynaptic sites of the individual neuron populations with respect to each other and the identification of putative synaptic partners using GRASP reenforce the hypothesis of synaptic interactions within the AME between dopaminergic/ serotonergic neurons and the PDF neurons. To shed light on the synaptic partners I performed first steps in array tomography, as it allows terrific informative analyses of fluorescent signals on an ultrastructural level. Therefore, I tested different ways of sample preparation in order to achieve and optimize fluorescent signals on 100 nm thin tissue sections and I made overlays with electron microscopic images. Furthermore, I made assumptions about synaptic modulations within the neuronal clock network via glial cells. I detected their cell bodies in close vicinity to the AME and PDFcontaining clock neurons. It has already been shown that glial cells modulate the release of PDF from s-LNvs’ terminals within the dorsal brain. On an anatomical level this modulation appears to exist also within the AME, as synaptic contacts that involve PDF-positive dendritic terminals are embedded into glial fibers. Intriguingly, these postsynaptic PDF fibers are often VIIAbstract part of dyadic or even multiple-contact sites in opposite to prolonged presynaptic active zonesimplicating complex neuronal interactions within the AME. To unravel possible mechanisms of such synaptic arrangements, I tried to localize the ABC transporter White. Its presence within glial cells would indicate a recycling mechanism of transmitted amines which allows their fast re-provision. Taken together, synapses accompanied by glial cells appear to be a common arrangement within the AME to regulate circadian behavior. The complexity of mechanisms that contribute in modulation of circadian information is reflected by the complex diversity of synaptic arrangements that involves obviously several types of neuron populations
All living organisms need timekeeping mechanisms to track and anticipate cyclic changes in their environment. The ability to prepare for and respond to daily and seasonal changes is endowed by circadian clocks. The systemic features and molecular mechanisms that drive circadian rhythmicity are highly conserved across kingdoms. Therefore, Drosophila melanogaster with its relatively small brain (ca. 135.000 neurons) and the outstanding genetic tools that are available, is a perfect model to investigate the properties and relevance of the circadian system in a complex, but yet comprehensible organism.
The last 50 years of chronobiological research in the fruit fly resulted in a deep understanding of the molecular machinery that drives circadian rhythmicity, and various histological studies revealed the neural substrate of the circadian system. However, a detailed neuroanatomical and physiological description on the single-cell level has still to be acquired. Thus, I employed a multicolor labeling approach to characterize the clock network of Drosophila melanogaster with single-cell resolution and additionally investigated the putative in- and output sites of selected neurons.
To further study the functional hierarchy within the clock network and to monitor the “ticking clock“ over the course of several circadian cycles, I established a method, which allows us to follow the accumulation and degradation of the core clock genes in living brain explants by the means of bioluminescence imaging of single-cells.
Eine intrauterine Gestationsdiabetes (GDM) Exposition induziert in den betroffenen Nachkommen eine lebenslang erhöhte Prädisposition für metabolische und komplexe Erkrankungen. Die Krankheitssuszeptibilität wird dabei durch epigenetische Veränderungen vermittelt, die sich über die Regulation der Genaktivität auch auf das Expressionsniveau und den Phänotypen auswirken. Um neue Gene zu finden, die eine Rolle in der fetalen Programmierung spielen, wurden in dieser Arbeit genomweite Methylierungsmuster von Nabelschnurbluten (FCBs) aus GDM-Schwangerschaften und Kontrollen miteinander verglichen. Mit Illumina Infinium HumanMethylation 450K Arrays konnten signifikante Gruppenunterschiede für insgesamt 65 CpG-Stellen (52 davon genassoziiert) festgestellt werden, die multiplem Testen standhielten. Mittels Pyrosequenzierung wurden vier dieser Kandidaten-Loci (ATP5A1, MFAP4, PRKCH, SLC17A4), sowie ein Gen aus der Literatur (HIF3A) genauer untersucht und die Effekte erfolgreich validiert. Für das zugrundeliegende multivariate Regressionsmodell wurden die potenziellen Störfaktoren Gestationsalter, kindliches Geschlecht und mütterlicher BMI berücksichtigt. Der GDM-Effekt zeigte sich stärker in der insulinbehandelten Subgruppe (I-GDM) als in der diätisch behandelten (D GDM) und scheint insgesamt multifaktoriell bedingt zu sein, da viele Gene betroffen waren, jedoch alle mit einer vergleichsweise niedrigen Effekt-Größe. Zusätzlich konnten für den MEG3 Promotor, MEST und PEG3, drei von vier geprägten Genen, die mittels Deep Bisulfite Sequencings (DBS) analysiert wurden, ebenfalls signifikante Methylierungs-unterschiede zwischen der GDM- und Kontroll-Gruppe detektiert werden. Die identifizierten Gene stellen labile Zielregionen für die GDM-induzierte intrauterine Programmierung dar und können zukünftig nützliche Biomarker für Krankheitsdiagnosen und Prognosen sein. Mittels DBS können darüber hinaus Einzelmolekül-Analysen durchgeführt werden, für die in differentiell methylierten Regionen (DMRs) anhand eines informativen SNPs die parentale Allel-Herkunft bestimmt und bei der Berechnung von Epimutationsraten einbezogen werden kann. Epimutationen wurde als solche gewertet, wenn sie ein > 50 % abnormal (de)methyliertes Methylierungsprofil aufwiesen. Die DBS-Daten wurden mit zwei verschiedenen Sequenzierplattformen generiert (Roche GS Junior und Illumina MiSeq). Für Zweitere wurde ein eigenes, unabhängiges Library-Präparations-Protokoll entwickelt. In Nabelschnurblut, adultem Blut und Viszeralfett wurden für die paternal exprimierte MEST Promotor DMR und die maternal exprimierte MEG3 intergenic (IG) DMR hohe Epimutationsraten für das jeweils unmethylierte Allel detektiert. Die geprägten (methylierten) Allele wiesen dagegen nur niedrige Epimutationsraten auf. Da MEST und MEG3 invers geprägte Gene sind, war die Hypermethylierung des nicht geprägten Allels (HNA) demnach unabhängig von der parentalen Allel-Herkunft. Die HNA scheint außerdem erst nach der Fertilisation aufzutreten, da in Spermien nur sehr wenige Epimutationen gefunden wurden. Für die sekundäre MEG3 Promotor DMR (deren Prägung von der primären MEG3 IG-DMR reguliert wird) wurde ein deutlich schwächerer, wenngleich signifikanter HNA-Effekt im FCB gemessen, für die paternal exprimierte PEG3 Promotor DMR konnte dagegen kein signifikanter Unterschied zwischen den beiden parentalen Epimutationsraten festgestellt werden. Der HNA-Effekt für die MEST DMR, MEG3 IG-DMR und MEG3 Promotor DMR war weder mit GDM noch mit Adipositas assoziiert und zeigte allgemein eine große interindividuelle Varianz. Die Aufrechterhaltung differenzieller Methylierungsmuster in Imprinting Kontrollregionen (ICRs) scheint in manchen Entwicklungs-Zeitspannen von großer Bedeutung und damit streng kontrolliert zu sein, später jedoch redundant zu werden, was sich in der Anreicherung von stochastischen sowie umweltinduzierten Fehlern auf dem nicht geprägten Allel äußern kann. HNA-suszeptible geprägte Gene ähneln in mancherlei Hinsicht metastabilen Epiallelen. Diese Studie zeigt, dass sowohl stochastische Faktoren als auch Umweltstimuli während der frühen embryonalen Entwicklung u.a. über HNA-Effekte geprägte Gen-Netzwerke programmieren, die in Wachstumsprozesse involviert sind. Um tiefere Einblicke in allelspezifische Prägungsprofile zu erhalten, wären umfangreiche DBS HNA-Längsschnittstudien aller 50-100 human geprägten Gene in unterschiedlichen Gewebetypen und Differenzierungsstadien wünschenswert.
Altersassoziierte und strahleninduzierte Veränderungen des genomweiten DNA-Methylierungs-Profils
(2018)
Der Prozess des Alterns ist ein komplexer multifaktorieller Vorgang, der durch eine sukzessive Verschlechterung der physiologischen Funktionen charakterisiert ist. Ein hohes Alter ist der Hauptrisikofaktor für die meisten Krankheiten, einschließlich Krebs und Herz-Kreislauf-Erkrankungen. Das Verständnis der epigenetischen Mechanismen, die in den Prozess des Alterns involviert sind, könnte zur Entwicklung pharmakologischer Interventionen beitragen, die nicht nur die Lebenserwartung erhöhen, sondern auch den Beginn des altersassoziierten funktionellen Abbaus verzögern könnten. Durch die Langzeit-Kultivierung primärer humaner Fibroblasten wurde ein in vitro Modell für das Altern etabliert, das die Identifizierung altersassoziierter DNA-Methylierungs-Veränderungen ermöglichte. Die in vitro Alterung konnte mit einer globalen Hypomethylierung und einer erhöhten DNA-Methylierung der ribosomalen DNA assoziiert werden. Darüber hinaus konnten DNA-Methylierungs-Veränderungen in Genen und Signalwegen, die für das Altern relevant sind, und ein erhöhtes epigenetisches Alter nachgewiesen werden.
Das in vitro Modell für das Altern wurde verwendet, um neben den direkten Effekten ionisierender Strahlung auf die DNA-Methylierung auch deren Langzeit-Effekte zu untersuchen. Die Strahlentherapie ist ein entscheidendes Element der Krebstherapie, hat aber auch negative Auswirkungen und kann unter anderem das Risiko für die Entwicklung eines Zweittumors erhöhen. Bei externer Bestrahlung wird neben dem Tumor auch gesundes Gewebe ionisierender Strahlung ausgesetzt. Daher ist es wichtig zu untersuchen, wie Zellen mit intakten DNA-Reparatur-Mechanismen und funktionierenden Zellzyklus-Checkpoints durch diese beeinflusst werden. In der frühen Phase der DNA-Schadensantwort auf Bestrahlung wurden in normalen Zellen keine wesentlichen DNA-Methylierungs-Veränderungen beobachtet. Mehrere Populations-Verdoppelungen nach Strahlenexposition konnten dagegen eine globale Hypomethylierung, eine erhöhte DNA-Methylierung der ribosomalen DNA und ein erhöhtes epigenetisches Alter detektiert werden. Des Weiteren zeigten Gene und Signalwege, die mit Krebs in Verbindung gebracht wurden, Veränderungen in der DNA-Methylierung. Als Langzeit-Effekte ionisierender Strahlung traten somit die mit der in vitro Alterung assoziierten DNA-Methylierungs-Veränderungen verstärkt auf und ein epigenetisches Muster, das stark an das DNA-Methylierungs-Profil von Tumorzellen erinnert, entstand. Man geht davon aus, dass Veränderungen der DNA-Methylierung eine aktive Rolle in der Entwicklung eines Tumors spielen. Die durch ionisierende Strahlung induzierten DNA-Methylierungs-Veränderungen in normalen Zellen könnten demnach in die Krebsentstehung nach Strahlenexposition involviert sein und zu dem sekundären Krebsrisiko nach Strahlentherapie beitragen. Es ist bekannt, dass Patienten unterschiedlich auf therapeutische Bestrahlung reagieren. Die Ergebnisse dieser Arbeit weisen darauf hin, dass die individuelle Sensitivität gegenüber ionisierender Strahlung auch auf epigenetischer Ebene beobachtet werden kann.
In einem zweiten Projekt wurden Gesamtblutproben von Patienten mit Werner-Syndrom, einer segmental progeroiden Erkrankung, und gesunden Kontrollen analysiert, um mit dem vorzeitigen Altern in Verbindung stehende DNA-Methylierungs-Veränderungen zu identifizieren. Werner-Syndrom konnte nicht mit einer globalen Hypomethylierung, jedoch mit einer erhöhten DNA-Methylierung der ribosomalen DNA und einem erhöhten epigenetischen Alter assoziiert werden. Das vorzeitige Altern geht demzufolge mit spezifischen epigenetischen Veränderungen einher, die eine Beschleunigung der mit dem normalen Altern auftretenden DNA-Methylierungs-Veränderungen darstellen.
Im Rahmen dieser Arbeit konnte die Bedeutung epigenetischer Mechanismen im Prozess des Alterns hervorgehoben werden und gezeigt werden, dass sowohl exogene Faktoren, wie ionisierende Strahlung, als auch endogene Faktoren, wie das in Werner-Syndrom-Patienten mutiert vorliegende WRN-Gen, altersassoziierte DNA-Methylierungs-Veränderungen beeinflussen können.