Transcription and DNA damage dynamics in the dopaminergic system following low dose ionizing radiation.

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Transcription and DNA damage

dynamics in the dopaminergic system

following low dose ionizing radiation.

P.G Mastroberardino, C. Milanese, S. Gabriels, L. Vermeer

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Information

Title: Transcription and DNA damage dynamics in the dopaminergic system following low dose ionizing

radiation.

Institution: Erasmus MC, Rotterdam

Address: Doctor molewaterplein 50-60, Rotterdam Department: Genetics, 7th floor

Supervisor Erasmus MC: P.G. Mastroberardino Supervisor Avans: A. Lindenbergh

School: University of applied sciences Avans Breda Address: Lovendijkstraat 61-62 Breda

Study: Biologisch en Medisch laboratorium onderzoek Name student: Leander Vermeer

Student nr: 2041554

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Abstract

During Parkinsons Disease, post-mitotic dopaminergic neurons degenerate after aging. The loss of dopaminergic neurons cause severe motor dysfunction, resting tremors, slow movement, rigidity and postural instability. Post-mitotic dopaminergic neurons do not tend to proliferate, however earlier studies showed that dopaminergic do repair damaged DNA. The precise mechanism of repair and its dynamics are still unclear. In this study we try to make clear what reactions Low Doses of Ionizing Radiation (LDIR) trigger in normal functioning mice. In this research we show that after induction of DNA damage by LDIR triggers MAPK responses which counteract the stress symptoms in the DA neurons. After time DA neurons return back in homeostasis. The findings in this research pave the way for further studies on the dopaminergic system, with for instance mice that elicit PD symptoms.

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1. Introduction

The research discussed in this report is commissioned by the Department of Genetics of the Erasmus Medical Centre in Rotterdam. The team of Dr. Mastroberardino mainly focusses on the molecular mechanisms underlying the pathogenesis of neurodegenerative disorders, and of Parkinson’s disease (PD) in particular. PD causes degeneration of a specific subclass of neurons that uses dopamine as a neurotransmitter – dopaminergic neurons - and that are located in the substantia nigra pars compacta region of the brain. Loss of dopaminergic neurons causes severe motor dysfunction and results in symptoms that include resting tremor, slow movements (or bradykinesia), rigidity, and postural instability. The research in this report investigated the role of the molecular processes responsible for maintenance of DNA quality and thus fidelity of the genetic information in the pathogenesis of PD. The studies are performed in vivo, in mouse models of PD, which recapitulate the essential features of the human disease.

Degradation of dopaminergic neurons involves multiple processes; compelling evidence indicates that increased levels of oxidation, and consequent accumulation of macromolecular damage in DNA and proteins, are major events in the progression of the pathogenesis. In agreement with this concept, DNA damage and increased levels of oxidation are induced by low doses of ionizing radiation.

The exact mechanisms underlying the detrimental effects of oxidation and DNA damage in PD, however, are still unclear. This research shows diverse results of transcription and DNA repair in healthy animals. The goal of this project was better understanding of the transcription and DNA repair dynamics in the dopaminergic system. Which is in its turn important for the research on PD pathogenesis.

We tried to detect DNA-repair in the brain after inducing damage by irradiation by immunohistochemical methods. The specific method used in this research was incorporation of Bromodeoxyuridine (BrdU) in the DNA during its repair. BrdU is a thymidine analog, which incorporates in the DNA during its synthesis, which can occur during replication or repair. After incorporation, it is possible to trace incorporated BrdU at cytological level, by immunological methods. To focus our studies on DNA repair in dopaminergic neurons and to exclude DNA replication, which in the adult brain occurs only in neurogenic cell population, we used multiple markers allowing unequivocal identification of non-neurogenic dopaminergic neurons. These compounds used are discussed in detail in chapter two.

In the following section we will discuss the background information of the disease, the techniques, and the chemicals used in the study. In chapter three, the experimental design will be presented.

Subsequently the materials and methods are displayed in chapter four, followed by the results in chapter five and the discussion in chapter 6.

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2. Theoretical background

2.1 Parkinson’s disease

Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease. Epidemiological studies estimate that the number of individuals with PD over age 50 in the most populous nations was in the magnitude of 4 million in 2005. Projections indicate that such figures will double to more than 8 million by 2030; PD is therefore a problem of high social relevance. No cures are available thus far, and therapies are limited to palliation of symptoms [12].

PD results in selective degeneration of a specific subclass of neurons, which use dopamine as

neurotransmitter: the dopaminergic (DA) neurons. Additionally, degeneration interests only those DA neurons in a particular anatomical domain of the brain, the Substantia Nigra Pars Compacta (SNpc), while those in the Ventral tegmental area are spared. The SNpc is part of the basal ganglia circuits, which also contain the striatum, the globus pallidus, and the subthalamic nucleus. These domains of the brain have important roles in motor control of the body and DA neurons in the SNpc exert inhibitory effects. When a large fraction (about 60%, figure 1) of these neurons degenerate, for instance in Parkinson’s disease, movement will be negatively affected and results in PD primary symptoms, which include resting tremor, stiffness, bradykinesia (i.e.) slower movement, and difficulty in walking [1].

Figure 1 cell loss time course between idiopathic and post encephalitic PD. At a loss of about 60 % of the DA neurons, symptoms start to show (from Agid, The Lancet…).

In most cases (i.e. >90%), the causes of PD are unknown and not attributable to monogenic mutations, and are therefore idiopathic. Only about 5% of the PD can be ascribed to single gene mutations; among the 18 loci assigned thus far, two genes (SNCA and LRRK2) have been conclusively associated with autosomal dominant and four with recessive PD (parkin, PINK1, DJ-1, and ATP13A2). The remaining vast majority of cases are sporadic and idiopathic (typical PD); here, etiology is likely to be due to complex synergistic interactions between environmental factors and predisposing genotypes. Genetic

susceptibility is associated with polymorphisms – rather than specific mutations - in LRRK2 and SNCA, such as the REP1 variants in the SNCA promoter region; additional predisposing factors are variants in the glucocerebrosidase (GBA) gene [16][14].

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Despite their mutation results in common clinical symptoms, genes associated with PD have very different functions. For instance, the biology of two of the major PD-associated genes, SNCA and LRRK2, is very distant. SNCA encodes for the protein α-synuclein, the key component of Lewy body inclusions, which are a hallmark of PD. The exact function of α-synuclein is still debated, but several indications suggest a role in synapse function. Linkage analysis studies have identified that PD- associated mutations in SNCA include dominantly inherited substitutions and genomic multiplications in familial, late onset Lewy body PD. Additional SNCA copies lead to earlier onset and more fulminant lewy body disease featuring dementia.

LRRK2 has GTPase and kinase functions. It also has multiple biological roles in striatal neurotransmission, neuronal arborization, endocytosis, autophagy and immunity. Patients with dominantly inherited mutations in this gene have a clinical phenotype that closely resembles idiopathic PD, yet not all the described mutations lead to Parkinsonism and only constitute a predisposing factor [14]. Autopsy on patients with LRRK2 Parkinsonism showed that typically Lewy body or neurofibrillary tangle pathology is present, leading to neuronal loss and gliosis. While genome-wide association studies (GWAS)

consistently confirmed LRRK2 as a PD-associated locus, they also highlighted that genetic variability in this gene can also to healthy aging of the basal ganglia. Further studies to clarify the role of LRRK2 variants in health and disease are warranted.

The vast majority of PD cases are idiopathic; here, etio-pathogenesis is complex and interests several cellular functions such as protein quality control, oxido-reductive homeostasis, and mitochondrial bioenergetics. In particular, epidemiological and laboratory studies associated toxins inhibiting mitochondrial respiration and complex I function with PD [14].

In both idiopathic and genetic PD cases, DA degeneration has been also associated with increased production of reactive oxygen and nitrogen species and augmented levels of oxidation [7]. Finally, PD pathogenesis is characterized by the formation of intraneuronal proteinaceous aggregates, the Lewy bodies (LB), which are mostly composed of the protein alpha-synuclein. Importantly, these aggregates are true hallmark of PD and are present also in those genetic cases that are caused by mutations in other than genes than alpha-syn (e.g. PINK) as well as in idiopathic cases.[13]

In the next sections we will discuss in greater details the process involved in our study, oxidative stress and its consequences on biological molecules.

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2.1.1 Oxidative stress in Parkinson’s Disease

Cellular metabolism is intrinsically associated with oxido-reductive reactions, in which biomolecules exchange electrons. The most typical case of such redox processes is mitochondrial respiration, which results in reduction of water to oxygen. The use of oxygen in this vital process, however, renders cells also intrinsically keen to oxidation because O2 is a good acceptor of electrons, which thus oxidizes other biological molecules. This oxidative tendency is counteracted by antioxidant systems, which provide additional electrons to maintain homeostasis. These systems are mostly based on thiol groups of cysteine (cys) residues. The unique chemical properties of thiols cysteine makes it extremely reactive towards reactive oxygen species (ROS) and thiol groups buffer the oxidation in the cell by undergoing an oxidative condensation to form disulfides. The reversible oxidation of protein thiols also performs a fundamental regulatory function because it acts as a molecular switch, which is mechanistically comparable to phosphorylation. In this switch protein activity is modulated through oxidation or reduction of thiols in critical positions. The thiol oxidation is therefore the major mechanism between ROS and signaling.

Differences in redox homeostasis between dopaminergic neurons and cortical neurons have been observed. DA neurons show a significantly higher level of oxidation while exposed to Rotenone, which possibly points out the presence of oxidative stress. The biology of these cells is interesting because the physiology is intrinsically associated with elevated ROS production. Most neurons use Na+_{to generate} their action potentials, but SNpc DA neurons rely on Ca2+_{. Over time the spontaneous activity of these} neurons could lead to elevated and harmful concentrations of Ca2+_{. This harm can be prevented by} buffering the activity of some organelles, such as mitochondria. The buffering activity is associated with the ROS production. In addition, excess ROS production could be derived directly from DA neuron metabolism. This production also generates harmful by-products such as semiquinones and hydrogen peroxide. The overall result is an increase in basal levels of ROS, which leads to further toleration of oxidative insults. This then leads to selective damage in the dopaminergic system and pro-oxidants such as rotenone and paraquat successfully mimic PD pathogenesis. Damage in the dopaminergic system is associated with pathogenesis of PD [7]

2.2 Exogenous sources of oxidative stress.

These molecules are charachterized by their ability of promoting the generation of reactive pro-oxidant species that shift the oxido-reductive homeostasis of the cell and lead to oxidative stress. Oxidative stress results in permanent and often detrimental modifications in biomolecules, including DNA and RNA. Oxidative stress therefore compromises cellular functions and affects the fidelity of the genetic information.

2.2.1. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)

Among the several neurotoxins damaging the basal ganglia or the substantia nigra and eliciting features of PD, MPTP is the most specific chemical in targeting neurons because intraneuronal uptake of import

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of its toxic metabolite is mediated by Dopamine Transporters. MPTP was discovered after drug abusers used this chemical while trying to synthesize a compound relating to the narcotic meperidine [17]. After self-injection of MPTP, the individuals developed movement disorders resembling PD. MPTP induces most of the biochemical, pathological and clinical features of PD in vivo and is one of the most used chemicals for providing animal PD models.

MPTP enters the brain cells easily through the blood-brain barrier, because it is lipid soluble. . MPTP is amphiphilic, so it will be captured in acidic organelles like lysosomes of astrocytes. MPTP itself is not toxic, but its oxidized form, 1-methyl-4-phenylpyridinium (MPP+_{) is. The astrocytes and serotonergic} neurons in the brain cells contain MAO-B, this compound oxidizes MPTP to MPP+_{. MPP}+_{reaches the} extracellular fluid and will be transported to the dopamine nerve terminals. In figure 2 a schematic representation of this mechanism is presented.

Figure 2 schematic representation of MPTP incorporation in the dopaminergic neuron

MPP+_{is an inhibitor of mitochondrial respiration, and in particular at the level of complex I [18]. Complex} I is the first enzyme in the mitochondrial electron transport chain and catalyzes the transfer of electrons to coenzyme Q. MPP+_{binds to the ubiquinone binding site and blocks enzymatic activity. Mitochondrial} derangement impairs ATP formation in the cell resulting in the inhibition of energy dependent processes, but also buffering of calcium by these organelles. Disruption of calcium ion homeostasis has a very important role in the neurotoxicity because increased levels of intracellular Ca2+_{are in general} detrimental leading to the activation of protein kinase and calpains I and II, which are Ca2+_regulated enzymes. These enzymes will facilitate neuronal death. Second, MPP+_{increases occurrence of oxidative} stress. Overall, these events contribute to the loss of dopaminergic neurons in PD. Some fundamental changes found in PD are not represented in MPTP exposure, like the Lewy-bodies. However it remains the most widely used chemical to model PD in laboratory animals [3].

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2.2.2. Pro-oxidant toxins

2.2.2.1 Paraquat (PQ)

PQ is a molecule with a similar chemical structure to MPTP (figure 3). PQ can be internalized in cells independently from DA transporters and therefore causes systemic intoxication not confined to DA neurons. When administrated in low doses, however, PQ causes detrimental consequences only in DA neurons of the SNpc [8]. PQ is a pesticide, used to spray rice fields to protect from pests, and is an environmental risk factor for PD. Paraquat replicates different aspects of the pathogenesis of PD.

Figure 3 molecule structures of Paraquat and MPP+. The small difference is that paraquat as an extra H3C group.

DA neurons seem to be sensitive for PQ after studies showed a significant decrease of DA neurons in the SNpc after intraperitioneal injections with PQ. On the other hand, NissI-stained neurons and GABAergic cells showed no decrease while threated with the same amount of PQ [8].

2.2.2.2. Rotenone

Another pesticide and pro-oxidant associated with PD pathogenesis is rotenone. Rotenone is an organic pesticide, which is found in the roots of some plants to avoid pest infestations. Rotenone is a specific complex I inhibitor, which has been associated with PD by epidemiological and laboratory studies [19] As MPP+_{, rotenone attaches to the ubiquinone binding site and there works as an inhibitor. Chronic}

exposure to rotenone in vivo reproduces many more features of PD including development of α-synuclein positive inclusions in nigral neurons, oxidative stress and apoptosis. Moderate levels of pro-oxidants such as rotenone and paraquat are well tolerated by organisms, but they still induce DA degeneration typical of PD. [10]

2.2.3. Irradiation

Another source of oxidative damage comes from irradiation. Exposure of the brain to low doses ionizing radiation in utero or during childhood, for instance in victims of nuclear accidents or oncological

pediatric patients, can lead to long-term damages and might contribute to neurological conditions in adulthood. Completed studies on atomic bomb survivors have in fact shown increased amount of neurological defects, microcephaly, and an increased frequency of seizures. These individuals

experienced prenatal exposure to ionizing radiation, in a developmental phase during which the brain is still growing. Also young children exposed to head irradiation or computed tomography for therapeutic reasons, for instance pediatric tumors, for which at present the cure has a very high rate of success, might develop neurological problems at later stages in life, as undesired side effects of the treatment [6].

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However, the exact consequences at a molecular level of low dose ionizing radiation exposure on the brain are poorly understood. A possibility is that radiation causes persistent impairment of mitochondria, also by inducing mutations and deletions in the mitochondrial DNA (mtDNA). mtDNA encodes for proteins that are essential for mitochondrial function and the loss or mutation of these genes can lead to dysfunction and disease. Cellular mechanisms for DNA repair in mitochondria are not as efficient as in other organelles, one of the reasons is that nuclear DNA repair mechanisms have a control mechanism which checks if the repair process is completed. Unraveling the effects of ionizing radiation in the brain is one of the topics of this research. [2].

This opens questions because while it is well ascertained that chemical pro-oxidants may cause selective degeneration of DA neurons, evidence on the effects of irradiation on this neuronal subtype is still unknown

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2.3 Methods to investigate alteration in nucleic acid metabolism.

2.3.1 BrdU (Bromodeoxyuridine)

Bromodeoxyuridine (BrdU) is a synthetic nucleoside and an analog of thymidine. During proliferation of cells or DNA repair the BrdU incorporates into the newly synthetized strands. The BrdU will be

incorporated instead of the normal thymidine nucleotide and will pair Adenine residues [4]. The BrdU molecule share the same fundamental structure as Thymidine, with a single substitution replacing a Methyl group with a Bromine (figure 4)

Figure 4 Left: Thymidine, Right: BrdU. The difference between thymidine and the thymidine analog (BrdU) is indicated by the red Br. Instead of the H3C a Bromine is present.

BrdU has been mostly used to detect proliferation in living tissues. The BrdU incorporates during the S-phase of the cell cycle, which is part of the replication of a cell. When further replication occurs, BrdU will be passed on from mother to daughter cell. Incorporated BrdU can be detected by immunological methods because specific antibodies recognizing these molecules have been developed. It has been proved that BrdU is detectable two years after infusion [5]. An example of detection of DNA replication with BrdU is shown in figure 5.

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Figure 5 DNA replication during female mitotic metaphase. Labeled with BrdU and FITC-immunodetection (green).a) Here is seen that replication starts at the end of the chromosome. b) BrdU labeled replication in the middle. c) BrdU labeled replication at a late period

In later studies they found out that BrdU also incorporates in cells which are being repaired, because of DNA damage. This DNA damage can be caused by ionizing radiation or neurodegenerative diseases for example. It is interesting to do research on neurodegenerative diseases like PD and Alzheimer’s disease, because the mechanisms of these diseases remain unknown. With the incorporation of BrdU in

damaged cells involved in these diseases it is possible to detect the BrdU and following the mechanism [6].

2.3.2 EDU (5-ethynyl-2’-deoxyuridine)

Although [3_{H] thymidine and BrdU labeling methods have been very useful for studying cell cycle} kinetics, DNA replication, DNA repair and assessing cell proliferation in normal or pathological cells, these methods have several limitations. Working with [3_{H] thymidine is inconvenient because of the} radioactivity. Autoradiography is also labor-sensitive and slow, because detection of [3_{H] thymidine can} take several months. Microscopic images of [3_{H] thymidine labeled cells have a poor resolution and low} signal-to-noise ratios. In comparison to [3_{H] thymidine labeling, BrdU immunostaining is faster and allows} microscopic imaging. One disadvantage of BrdU staining is that the complementary base pairing blocks the access of the anti-BrdU antibody (AB) to BrdU subunits. The exposure of the BrdU epitope involves strong denaturing conditions to the cell and tissue samples such as concentrated hydrochloric acid or mixtures with methanol and acetic acid. These staining conditions possibly degrade the structure of the specimen and also make the intensity of the BrdU staining highly dependent on the conditions used by each investigator. As with any immunohistological stain, the size of the tissue piece to be stained is limited by the penetration of the AB through the fixed tissue. This limitation can be overcome with sectioning or by using long incubation times.

EdU(5-ethynyl-2’-deoxyuridine) is a new alternative for BrdU assay to directly measure active DNA synthesis or S-phase synthesis in the cell cycle. EdU is also a nucleoside analog of thymidine and is incorporated in the DNA during active DNA synthesis in place of thymidine. Detection of EdU is based on

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click-it chemistry, a reaction between an azide and an alkyne. The Edu contains an alkyne functional group, which reacts with azide- containing reagents to form a stable triazole ring (figure 6).

Figure 6 Left: incorporation of EdU in DNA backbone. Right: reaction of fluorescent dye with EdU for detection.

The advantages of using the EdU click reaction compared to traditional BrdU staining are wide ranged. First of all the click reaction is highly specific and relatively fast because it requires only a minimal number of steps. The fluorescence signal is strong so detection is easier. The conditions in which the reaction takes place are non-denaturing and mild. The small fluorescence azide molecules will easily enter cells in contrast to BrdU antibodies. Finally, click chemistry is very versatile because it is possible to use azide reagents conjugated with a large variety of fluorophores, or even biotinylated moieties for purification purposes [9].

2.3.3 EU (5-ethynyluridine)

5-ethynyluridine (EU) incorporation in RNA is a method to detect RNA synthesis in a cell. When RNA in a cell is formed, this means that transcription of the DNA is in progress. EU is the associated with the earlier discussed EdU which incorporates into DNA synthesis. The detection of EU works the same as in EdU. Detection of incorporated EU into RNA is also done with click-it chemistry, which provides clear fluorescent signals. The EU does not label cellular DNA, making it an specific transcriptional label.[11]

2.3.8 The DNA double strand break marker H2Ax

γ

DNA damage can be categorized in two types: endogenous and exogenous. Endogenous DNA damage is inflicted by harmful chemical species produced by the cell, for instance reactive oxygen species, which are by-products of normal metabolic activity. Other pathways, such as apoptosis, or depurination, can also contribute to DNA damage. Exogenous DNA damage can be caused by exposure to physical damage such as radiation or chemical agents such as cytotoxic drugs. The inflicted DNA damage can lead to different kinds of lesions: base damage, sugar damage, single strand breaks (SSBs), and double strand breaks (DSBs). Of all damage types, DSBs are particularly toxic. Unrepaired DSBs are extremely

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detrimental for the cell and its consequences, such as losing the centromere-distal segment of the chromosome, are lethal. DNA damage induction by ionizing radiation mainly causes DSBs [28], so measuring and analyzing DSBs in neurons after exposure is of particular interest. Many methods have been used to detect DSBs, such as neutral elution, pulse field electrophoresis and comet assays, but monitoring the phosphorylation of the H2A histone family, member X (γ-H2AX) and formation of foci, which both occur as a consequence of DSB, is a particularly precise strategy. This method is highly sensitive and easier to perform than other techniques.

DNA is normally wrapped around a core histone molecule forming the nucleosome complex. Histone cores are built up by histone proteins called H2A, H2B, H3 and H4. The H2A protein has the greatest number of variants including the H2AX variant. In human cells H2AX accounts for approximately 10% of the H2A protein [29]. H2AX can be phosphorylated on Ser1, acetylated on Lys5, and ubiquitinated on lys 119; after DNA damage, it is phosphorylated on Ser139. The phosphorylated form of H2AX is called γ-H2AX, which is a key factor in repairing damaged DNA because it facilitates recruitment of other factors of the DNA repair machinery. When double strand breaks are formed, for instance as a consequence of ionizing radiation, hundreds of thousands of H2AX proteins are phosphorylated. Proteins within a few megabases of the damage site are involved and propagate to the site of damage [30][31].The fraction of H2AX that is phosphorylated depends from the extent of damage and is relatively constant for given doses; for instance, a 1Gy dose of X-rays induces phosphorylation of 1-2% of H2AX [30]. On the

methodological aspect, γ-H2AX foci – which can be detected by highly specific and validated antibodies - will form at the site of damage.. Because the relationship between H2AX phosphorylation and DSB is well characterized and immunodetection methods are highly sensitive, this method will be used further on as DNA damage markers and particularly for DSBs.

2.3.4 The gene silencing marker 5-Methylcytosine

5-methylcytosine (5mC) is the cytosine nucleotide modified by the addition of a methyl group to the 5th carbon (figure 6).

Figure 6 molecule structures of Cytosine and 5-methylcytosine. Note that the methylgroup(H3C) is added at the 5th _carbon group. The molecular weight increase with approximately 14Da.

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The methylation typically occurs at cytosine in CpG dinucleotides in vertebrates. Since CpG is a simple palindromic sequence, it pairs with another CpG sequence on the complimentary strand. If one of these cytosines is methylated, very often also methylation occurs also on the other one [20].

5mC is a critical repressor of transcription in the genome. When 5mC is present in promotors, it is associated with stable, long-term silencing. This silencing occurs by either blocking positive transcription factors or promoting the binding of negative factors. Several classes of proteins that facilitate

transcriptional repression recognize methyl-CpG sites, including Methyl CPG Binding Domain (MBD), SET and RING finger domain (SRA) and kaiso protein families.[21]

Because 5-methylcytosine is a repressor of transcription, a triple staining with anti 5-methylcytosine, TH and 53BP1 could give a more clear perspective on the transcriptional state of DA neurons after exposure to ionizing radiation.

2.3.5 The marker of active transcription histone H3 lysine 27 acetylation

(K27ac)

DNA is compacted, folded, and organized within chromatin in the nuclei of eukaryotic cells. The fundamental subunits of chromatin are nucleosomes. These nucleosomes form a chain of histone proteins around which DNA is circumscribed. The four histones H2A, H2B, H3, and H4 form the octamer center of the nucleosome. Biological programs are controlled by transcription factors and chromatin regulators, which maintain specific gene expression through epigenetic modifications in histones. These histones are subjected to a wide variety of modifications such as lysine and arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation.

Lysine 27 on histone 3 (H3K27) can be both methylated and acetylated. Former evidence suggested that H3K27 was associated with transcription silencing and, indeed, when H3K27 is trimethylated

(H3K27me3), it is closely associated with inactive gene promoters. Here, H3K27m3 serves as a signal for binding of a repressor complex of the polycomb family (PRC1), which blocks the recruitment of

transcriptional activation factors such as SWI/SNF and prevents initiation of transcription by RNA polymerase II. [34][35].

Differently than methylation, acetylation of H3K27 (H3K27ac) is highly enriched at promoter regions of transcriptionally active genes[36], including the genes which are targeted by polycombs. Acetylation occurs at the same sites of methylation that form H3K27m3, which is therefore prevented by H3K27ac. The presence of H3K27ac in neurons indicates genomic sites of active transcription, which code for genes necessary to cope with the stress induced by ionizing radiation. We will use both this marker and the transcription repressor 5meC to determine whether γ-H2AX foci that persist for long periods after damage induction are in actively transcribed or silent DNA regions.

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2.3.6 Glial fibrillary Acidic Protein (GFAP)

The nervous system is composed by neurons and glial cells. The relevance of glial cells for proper functioning of the brain has been receiving growing interest and is now clear that these cells do not play only support role, for instance as a source of trophic factors (e.g. BDNF), but are also involved in

important processes concerning metabolism, homeostatic regulation of neurotransmitters and ions in the intersynaptic space, synapse formation, and myelination [39]. In development of the brain, newly formed neurons are guided by glial cells to their final site of residence. After trauma or during chronic neurodegenerative diseases, glial cells become more active to counteract the detrimental processes. Glial activation, however, must be carefully controlled by the organism because when excessive, especially in microglia, is detrimental [40]

Glial Fibrillary Acidic Protein (GFAP) is a major constituent of glial intermediary filaments that form the cytoskeleton of mature astrocytes. Astrocytes are star shaped sub-type of glial cells in central nervous system. Astrocytes are fundamental for a correct development of brain architecture, are the most abundant glial cells in the brain, and are closely associated with neuronal synapses. Astrocytes are known to support neurons by providing nutrients such as lactate and glucose. To date it is well

recognized that glial cells are essential for the survival of DA neurons. Striatal Oligodentrocyte-type 2, for instance, are critical to ensure survival and correct phenotypic development of DA neurons in culture [32]. The density of GFAP-positive astrocytes appears to be inversely related to the magnitude of neuronal loss across the different main dopaminergic areas of the brain in PD post-mortem samples, therefore suggesting that areas with poor population of astrocytes are more prone to degeneration [33].

2.3.7 Cell cycle re-entry marker Ki67

In an attempt to visualize DNA repair in neurons, we used the thymidine analogue BrdU and followed its incorporation in cells outside the S-phase; in these condition, DNA synthesis could be ascribed to repair, rather than replication. To perform this study, however, it is essential to perform co-staining with multiple antibodies in which BrdU is paralleled by markers for cell cycle re-entry. In the mid-1990s, it was proposed that cell-cycle reactivation in post-mitotic neurons might have been a crucial step in neuronal loss in Alzheimer’s disease. Indeed, later studies showed that the brain of patients with

neurodegenerative disorders such as PD showed an increase in the expression of proteins involved in the cell cycle. Therefore, cell-cycle re-entry can be seen as a prelude to apoptotic cell death in neurons (37). Oxidative stress induced for instance by ionizing radiation or MPTP is associated with cell cycle re-entry and neuronal death [41]

To determine whether BrdU incorporation in DA neurons occurs because of repair or is rather caused by cell cycle re-entry, we monitored the latter using an antibody against the protein Ki-67. The expression of the human Ki-67 protein is strictly associated with cell proliferation. In 1993, the entire cDNA

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sequence coding for Ki-67 was published. It was expected that the identification of sequence motifs of potential significance and homology searches would lead to more knowledge of the actual function of the molecule. The primary structure revealed numerous interesting features. For example, two differently spliced mRNA isoform were found with sizes of 8688 and 9668 bp. The forms differ by the absence and presence of the region encoded by exon 7 of the Ki-67 gene. Also, a potential ATP/GTP binding site motif a loop was predicted in the carboxy terminal region. Even though some of the features predicted by the sequence analysis have been verified and characterized, the function of Ki-67 still remains unclear. The difficulties of determination of the function can be attributed to the absence of obvious homology with other proteins. The Ki-67 protein should be present in all active phases of the cell cycle. [38]. By processing this information, our hypothesis is when the Ki-67 protein is expressed in DA neurons, the cell has re-entered the cell cycle. BrdU and Ki-67 co-localization in DA neurons would indicate that the cell is not repairing, but it rather re-entered cell cycle to commit apoptosis. This can be determined by a multiple staining using simultaneously antibodies against TH, BrdU and Ki-67.

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3. Experimental design

Optimization of the methods

Detection of DNA damage in vivo

Initially, we will monitor DNA repair capacity by assessing BrdU incorporation. Because this compound has not been used to investigate DNA repair in PD before, the project involves substantial pilot

experiments to define the optimal technical conditions. A first challenge will be to determine BrdU dosage and timeline of administration to label neurons repairing DNA rather than other types of cells that can replicate (e.g. neurogenic cells). Initially, we will intraperitioneally inject mice with different concentrations of BrdU. To induce DNA damage, we will expose mice to a total body ionizing radiation with a 0.1, 0.5 and 2 Gy from a gamma source. Ionizing radiation is used because chemical consequences on DNA have been well characterized and it is known that it induces several types of DNA lesions [15] BrdU will incorporate in the DNA that is synthesized during damage repair. Low doses of ionizing

radiation cause persistent damage, such as impairment of cardiac mitochondria, where complex I and III are targeted. Ionizing radiation will also induce mutations and deletions in the mitochondrial DNA (mtDNA). The loss of these genes can lead to mitochondrial dysfunction and eventually to apoptosis.

Detection of transcription levels in vivo

EU incorporation is a method to detect RNA synthesis in a cell. If there is RNA synthesis in a cell there will be transcription within this cell. When there is DNA damage in a cell, transcription will be blocked. The goal of this step is to find out if there is a decrease in transcription in the cells when being exposed to irradiation as above. After exposure the mice will be injected intraperitioneal with EU, a modified precursor of RNA. Detection of EU will be done taking advantage of click-it chemistry, which has been explained in chapter 2.4. This method of detection has not been attempted in the brain thus far and requires pilot experiments to determine technical details, including EU dosage and time course. We will start following the indications described in the original paper: mice will be injected with 100 l of a 20 mg/ml solution of EU in PBS. Tissues will be collected 5 h after injection and fixed in formalin overnight [11].

DNA damage and transcription alteration in the brain after ionizing radiation

Different neuronal subtypes may also have very different repair properties, due to different sensitivity to DNA damage or a diverse repertoire of DNA repair mechanisms. For example some neuronal cells may initiate DNA repair much faster than other cells, which makes them less sensitive to DNA damage. We will therefore take advantage of triple immunofluorescent staining technique using antibodies against BrdU, an antibody against a marker for neurogenic cells, and antibodies against markers for specific types of neurons (i.e. Dopaminergic neurons). To identify DA neurons we will use a specific antibody against Tyrosine Hydroxylase, which is the rate-limiting enzyme in dopamine synthesis and catalyzes the reaction involving the amino-acid tyrosine, a building block of dopamine. This experiment will determine the best

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dose of BrdU to obtain a clear staining of DNA repair in damaged DA neurons, so we can proceed with the next steps in the project.

DNA damage and repair in DA neurons in vivo

Next, we will apply the information obtained in the previous experiments to study DNA repair in a healthy c57bl/6. Mice will be pre-treated with BrdU (0.2mg/g) by injecting them subcutaneously, and exposed to 0.1, 0.5 and 2Gy of ionizing radiation to induce DNA lesions. To see if damaged DA neurons incorporated with BrdU and γH2AX indicates DNA repair rather than apoptosis, combination with antibodies against k27ac(active transcription marker) and later Ki67(proliferation/apoptosis marker) is done. When K27ac, γH2AX and BrdU co-localize, this could possibly mean that these neurons are actively transcribing DNA repair genes. When Ki67, γH2AX and BrdU co-localize, this means that these cells go into S-phase and as earlier mentioned, in apoptosis.

Detection of gene silencing in DA neurons in vivo

5-methylcytosine is a methylated form of the DNA base cytosine and the expression of these methylated genes may be altered. At the fifth atom of the 6-atom ring, a methyl group is attached to the cytosine and makes it 5-methylcytosine. Different amounts of exposure to ionizing radiation may have different effects on the methylated state of the cytosine and thus to the expression of the genes involved. To determine which effect the ionizing radiation has on dopaminergic neurons and its DNA, a triple staining will be performed with antibodies for TH, anti 5-methylcytosine and 53BP1. To identify DA neurons we will use a specific antibody against Tyrosine Hydroxylase (TH). Methylated cytosine will be detected by anti 5-methylcytosine, which detects the methylated group on the cytosine. The 53BP1 and /or γH2AX antibody will be used to detect DNA damage, induced by the exposure to 2Gy of radiation.

Co-localization between 53BP1 and anti 5-methylcytosine will give more understanding of the methylated state of the cytosine base caused by the depleting effects of radiation. This experiment will be done of 4 and 24 week old mice to determine the difference in epigenetic changes.

Detection of active transcription in DA neurons.

The chemical consequences of ionizing radiation to DNA are well characterized, it induces several types of DNA lesions. In our interest is the brain of the mouse and especially DA neurons. To determine the amount and formation of active transcription regions of DA neurons exposed to LDIR, K27ac is used as marker. Acetylation of H3K27 (H3K27ac) is highly enriched at promoter regions of transcriptionally active genes. To determine the difference in active transcription after 4 and 24 weeks after irradiation, a staining is done with antibodies against TH, γH2AX and K27ac.

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4. Materials and methods

4.1 Pilot experiments for BrdU incorporation

Before using BrdU to monitor DNA repair capacity, there was need for substantial pilot experiments to define the optimal technical and practical conditions to use BrdU incorporation in brain tissue. 10 days old C57BL/6 mice were used and pre-treated with BrdU (0.2 mg/g). After BrdU treatment, the mice were exposed to 2 Gy of ionizing radiation to induce DNA damage. Two experiment groups and one control group were created to investigate the timeline of BrdU administration. The groups are displayed in table 1.

Table 1 Experiment group 1. Groups BrdU incorporation pilot experiment. First BrdU (0.2 mg/g) was injected subcutaneously into 10 day old c57Bl6 mice. In Group (a) a two hour waiting step was used for BrdU administration into the brain cells. After two hours the mice were exposed to 2 Gy of ionizing radiation and there was waited two hours for sacrificing. Group (b) was exposed to 2 Gy of ionizing radiation directly after injection with BrdU, again 2 hours was waited for sacrificing. Group(c) was used as control group. BrdU was injected, after this four hours was waited for sacrificing (no exposure to ionizing radiation)

Group BrdU incorporation Radiation A BrdU 2hr 2 Gy 2hr sacrifice B BrdU  2 Gy 2hr sacrifice C BrdU 2hr - 2hr sacrifice

After sacrificing, mice were perfused; this is a method to get all the remaining blood out of the body and brain tissue because any blood remaining in blood vessels can interfere with immunological detection. Perfusion is done by pumping a flushing solution through the heart, after this, a fixation solution will be pumped through the body to maintain organs and tissues.

4.1.1 Perfusion and gelatin embedding of tissues

Initially, the perfusion tube was flushed with flushing solution (50ml Phosphate Buffer (PB) 0.2M, 4.5g NaCl, 450 ml milli Q). Subsequently, mice were intraperitoneally injected with 150 µl pentobarbital to anesthetize them. After the pain reflex was gone, the chest of the mouse was opened, when the heart was reached the perfusion needle was inserted in the left ventricle. After this a small incision was made in the right atrium and the pump was turned on at a speed of 4ml/min for about 1-2 minutes until all blood was out of the body. Now the pump was switched to the fixing solution (4% PFA in milli Q), the fixation solution reaching the animal was seen by contraction of the muscles and the tail going up. At this point the animal was perfused for 5 minutes. The liver should have lost its red color and the animal has become stiff if the perfusion was successful. Now take out the tissues which were needed to be

embedded in gelatin or paraffin. Tissues should now be fixed in 4% PFA for 2 hours at 4°C.

For gelatin embedding a small layer of 12% gelatin/ 10% sucrose at 37°C was poured into a plastic embedding mold and let solidify. After this, the tissues were put on the layer of gelatin and embedded in 12% gelatin/ 10% sucrose and marked how the tissues are placed and the gelatin was solidified. After the gelatin was solid, the block was taken out of the mold and put in 30% sucrose/ 10% formaldehyde for 2 hours at room temperature. Now the gelatin blocks were put in 30% sucrose/ 0.1 M PB and left at 4°C

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overnight. After this the blocks were ready to be cut into sections and immunohistochemistry (IHC) could be performed on these sections.

4.1.2. BrdU pilot staining

For defining the optimal technical and practical conditions to detect BrdU in brain tissue, pilot stainings were done. For these pilot staining specific regions of the brain were stained: the subventricular zone and the olfactory bulb. These regions were carefully picked because of the high amount of proliferating neurons, so BrdU could incorporate in the DNA during this proliferation. After pilot staining on these regions, the pilot was done on the substantia nigra.

From the start of this pilot different protocols were used which led to an optimal protocol according to results obtained from these protocols. The following is the first protocol used during optimization of the staining on BrdU.

After selection of slides containing the subventricular zone and the olfactory bulb, the slides were washed 4 times 5 minutes with in PBS. Now it was incubated 30 minutes in 0.5% Triton X-100, followed by a 30 minute incubation in 0.1M HCl on ice. After this the slides were incubated 45 minutes in 2M HCl at 37°C, the incubations in triton and HCl were part of an antibody penetration improvement method. Now the slides were neutralized for 10 minutes in 0.1M sodium borate buffer (pH 8.5) at Room Temperature (RT). The next step was to block the samples, this was done by incubation in the blocking buffer (0.1M PBS, 0.5% Triton X-100 and 10% normal serum) for 60 minutes at RT. After this the first antibody (AB) for BrdU (Mouse, DAKO M0744) was incubated overnight at 4°C. Sections were now washed extensively 5 times 10 minutes in PBS. Incubation of the secondary AB (Cy3 goat anti-mouse, Jackson) was done in blocking buffer for 2 hours at RT. Again the sections were washed 5 times 10 minutes in PBS. Lastly the sections were put on slides by using chromaluin and were covered with vectashield.

4.2 Detection of DNA damage in vivo

Due to pilot results, a different staining protocol was used for the detection of DNA damage in vivo. This protocol is a combination between the BrdU pilot protocol and a protocol for standard

immunofluorescence with an antigen retrieval step.

Sections were carefully selected for the presence of the substantia nigra, groups used can be found in table 1. The slides were washed 4 times 5 minutes with in PBS. Now it was incubated 30 minutes in 0.5% Triton X-100, followed by a 30 minute incubation in 0.1M HCl on ice. After this, the slides were incubated 45 minutes in 2M HCl at 37°C, the incubations in Triton X-100 and HCl were part of an antibody

penetration improvement method. Now the slides were neutralized for 10 minutes in 0.1M sodium borate buffer (pH 8.5) at RT. After neutralization, washing was done 2 times 10 minutes in demineralized water. For antigen retrieval step, incubation in pre-heated sodium citrate buffer for 30 min at 80°C was necessary. After cooling down for 30 minutes it was washed with 2 times 5 minutes in demineralized water, following a 2 times 10 minute washing step in PBS. The next step was to block the samples, this was done by incubation in the blocking buffer (0.1M PBS, 0.5% Triton X-100 and 10% normal serum) for

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60 minutes at RT. After this the first antibodies for BrdU (Mouse, DAKO M0744, 1:100), TH (Sheep, Novus Biologicals 300-110, 1:4000) and 53BP1 (Rabbit, Santa Cruz 22760, 1:1000) were incubated overnight at 4°C. Sections were now washed extensively 5 times 10 minutes in PBS. Incubation of the secondary antibodies (all 1:500) for BrdU (Alexa 488, donkey anti-mouse), TH (Cy3, donkey anti-sheep) and 53BP1 (Alexa 647, donkey anti-rabbit) was done in blocking buffer for 2 hours at RT. Again the sections were washed 5 times 10 minutes in PBS. At last the sections were put on slides by using chromaluin and were covered with Vectashield. Confocal scanning microscopy was used to detect fluorescence.

4.2.1. Pilot staining sensitivity TH to HCl treatment

After earlier results showing low TH staining when using the antibody penetration improvement step, a pilot for defining sensitivity of TH staining for HCl was done. The protocol presented in chapter 4.2 was used, except when indicated otherwise. For this staining the substantia nigra was selected from the sections and differentiating groups were created (table 2). Also higher TH antibody concentrations were included.

Table 2 Experiment group 2. Groups pilot staining sensitivity TH/HCl on c57Bl6 mice brains. In group 1 and 2 a TH 1st_AB concentration of 1:2000 was used, Group 1 used HCl treatment and group 2 was stained without HCl treatment. In group 3 and 4 a TH 1st_{AB concentration of 1:4000 was used, Group 3 used HCl treatment and group 4 was stained without HCl} treatment.

Group Concentration TH 1st_AB _Protocol

1 1:2000 Protocol 4.2 with HCl treatment

2 1:2000 No HCl treatment

3 1:4000 Protocol 4.2 with HCl treatment

4 1:4000 No HCl treatment

4.2.2 Detection of DNA damage in vivo using a triple staining with TH, BrdU and 53BP1 The following protocol is the completely optimized method for performing a triple staining by using antibodies for TH, BrdU and 53BP1. This protocol will be used for further research and can be found in appendix 1.

Sections were carefully selected for the presence of the substantia nigra, groups used can be found in table 1. After selection of the sections a washing was done 4 times 10 minutes in PBS, followed by a blocking step by incubation 90 minutes in blocking buffer (0.1M PBS, 0.5% Triton X-100 and 10% normal serum) at 4°C. Subsequently the incubation with the first antibodies for TH (Sheep, Novus Biologicals 300-110, 1:500) and 53BP1 (Rabbit, Santa Cruz 22760, 1:1000) was done overnight at 4°C.

The next day was started by a washing 4 times 10 minutes in 0.1% PBS/Tween20 (PBST). Now the incubation with the secondary antibodies for TH and 53BP1, respectively Cy3 (TH) and Alexa 647 (53BP1) were incubated in blocking buffer for 2 hours at RT. The sections were now washed 4 times 10 minutes in PBST. For maintaining fluorescent signal of the detection of TH and 53BP1 because of later use of HCl, the section were fixed with 4% paraformaldehyde. Again the sections were washed 4 times 10 minutes in

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PBST. Denaturing the sections was done by incubation in 50% formamide in 2x SSC buffer (300mM NaCl, 30mM sodium citrate) for 90 minutes at 65°C, followed by 30 minutes in 2M HCl at 37°C. Denaturing is necessary so the BrdU AB can detect BrdU in the DNA. Due to highly acidic conditions, the sections were neutralized 2 times 5 minutes in sodium borate buffer (0.1M, pH 8.5). Again sections were washed 4 times 10 minutes in PBST. After incubating 90 minutes in blocking buffer, the sections were incubated overnight at 4°C with the primary AB (1:500) for BrdU in blocking buffer.

The sections were now washed 4 times 10 minutes in PBST. Incubation with the secondary AB for BrdU (Alexa 488, 1:500) was done for 2 hours at RT. Again the section were washed 4 times 10 minutes in PBST. At last the sections were put on slides by using chromaluin and were covered with Vectashield. Confocal scanning microscopy was used to detect fluorescence.

4.3 Detection of DNA transcription alteration in vivo using a triple staining

with TH, anti 5-methylcytosine and 53BP1

To detect altered DNA transcription when exposed to ionizing radiation, a triple staining was performed with antibodies for TH, anti 5-methylcytosine and 53BP1. For a pilot staining to demonstrate the anti-5-methylcytosine AB, the protocol displayed in appendix 1 was used. The triple staining was performed after good results from the pilot experiment. The protocol displayed in appendix 1 is used with the following specific content:

The only alteration was the replacement of the BrdU AB by the anti-5-methylcytosine (Millipore, MABE146, mouse). To identify co-localization in the DA neurons, the sections used were carefully selected for the presence of the substantia nigra. The groups of mice used are displayed in table 3. Table 3 Experiment group 3. All groups are C57bl6 mice collected 4 weeks after irradiation. Group 1 is a control group, which not have been irradiated. Group 2 is exposed to full body irradiation with 0.1 Gy. Group 3 is exposed to full body irradiation with 0.5 Gy. Group 4 is exposed to full body irradiation with 2 Gy.

Group Genotype Age Remarks

1 C57bl6 4 weeks Control

2 C57bl6 4 weeks 0.1 Gy

3 C57bl6 4 weeks 0.5 Gy

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4.4 Detection of DNA damage response in DA neurons

Earlier results showed no BrdU incorporation in DA neurons. A new triple staining was done with antibodies for BrdU, TH and 53BP1. Only a different protocol for injection and BrdU incorporation times compared to the experiment in chapter 4.2.2. is used. Two-hour incorporation of BrdU after irradiation showed to be too short, so longer incorporation times were used. The new experiment has 2, 4 and 6 hours of incorporation time before sacrificing of the mice was done (figure 16).

Figure 8 Figure Experiment group 4, schematic scheme BrdU incorporation using longer incorporation times. Approximately 10 day old c57bl/6 mice were used in this experiment. The scheme is divided into 2 groups: the control group (CTRL) and the experiment group (EXP). CTRL group is not irradiated, EXP group is irradiated. Mice were collected after 2(group A, D), 4(Group B, E) and 6(group C, F) hours. Mice were perfused and brain was dissected, embedded in gelatin and cut in sections.

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4.5 Detection of active transcription in DA neurons with incorporated BrdU

After longer incubation of BrdU in mice exposed to ionizing radiation, BrdU was seen incorporated in several DA neurons. To see if these DA neurons incorporated with BrdU are actively transcribing, a staining with markers against TH, BrdU and K27ac was used. As described in chapter 2.3.5., acetylated histone H3 (k27ac) is a marker for positive transcription and prevents trimethylation. Positive

transcription in a cell incubated with BrdU could support that the DA neurons are actually repairing. For this research, mice from experiment group 4 (figure 16) are used. The protocol displayed in appendix 1 is used. The specific content in this IF is presented in table 4.

Table 4 Specific content IF triple staining TH-BrdU-K27ac Content

Brain region Substantia Nigra

Experiment group Experiment group 3; table 3

1st_{primary AB} _{TH (sheep, Novus biologicals, 300-110, 1:500)} 1st_{secondary AB} _{Against TH: Cy3 (affinipure, 713-165-147, 1:500)}

2nd_{primary AB} _{BrdU (mouse, DAKO 347580, 1:500)}

K27ac (rabbit, AB-4729, 1:1000)

2nd_{secondary AB} _{Against BrdU: Alexa 488 (A21202,1:500)}

Against K27ac: Alexa 647 (A31372, 1:500)

4.6 Detection of glial cells with GFAP

As mentioned in chapter 2.3.6. , GFAP is a protein present in glial cells and especially astrocytes. These glial cells gives an insight on the supportive status of these cells when the DA neurons are exposed to the damaging ionizing radiation. Also, it is not sure what cell types the cells in vicinity of the DA neurons are, which also show presence of 5-mc. These cells could be either glial cells or another cell type. To

determine the amount of glial cells, an IF is done with antibodies for TH and GFAP. In this same IF also an antibody for 5-mc is used to determine if the glial cells are the 5-mc positive cells in the surrounding of the DA neurons. The sections used for this IF are from experiment group 3 (table 3). The protocol displayed appendix 1 is used. The specific content in this IF is presented in table 5.

Table 5 Specific content IF triple staining TH-GFAP-5mc Content

1st_{primary AB} _{TH (sheep, Novus biologicals, 300-110, 1:500)}

GFAP (rabbit, DAKO, 20334)

1st_{secondary AB} _{Against TH: Cy3 (affinipure, 713-165-147, 1:500)}

Against GFAP: alexa 647 (A31373, 1:500)

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2nd_{secondary AB} _{Against 5-mC: Alexa488 (A21202, 1:500)}

4.7 Detection of cell-cycle re-entry in DA neurons

BrdU incorporation was seen in DA neurons after different incorporation times. This was tested by using the experiment in chapter 4.4. Even BrdU positive DA neurons were found co-localized with k27ac, a marker for active transcription. Now, it was mandatory to check if the DA neurons incorporated with BrdU were actually repairing or re-entering the cell cycle. When re-entering the cell cycle, the DA neurons are going into programmed apoptosis. To determine this, an immunofluorescence (IF) staining was done on groups presented in figure 16. The IF staining will be done with antibodies against TH, BrdU and Ki-67. The protocol for this IF is displayed in appendix 1. The specific content in this IF is presented in table 6.

Table 6 Specific content IF triple staining TH-BrdU-Ki-67 Content

Experiment group Experiment group 4; Figure 16

1st_{primary AB} _{TH (sheep, Novus biologicals, 300-110, 1:500)} 1st_{secondary AB} _{Against TH: Cy3 (Affinipure, 713-165-147, 1:500)}

2nd_{primary AB} _{BrdU (mouse, DAKO 347580, 1:500)}

Ki-67 (rabbit, thermo scientific pa5-19562, 1:200)

2nd_{secondary AB} _{Against BrdU: Alexa 488 (A21202,1:500)}

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4.8 Detection of active transcription in 4week and 24 week old mice, exposed

to ionizing radiation

Ionizing radiation is said to have effect on transcription levels in DA neurons. To determine the effect of different doses of ionizing radiation on the active transcription levels at young mice (4 week after irradiation) compared to adult mice (24 week after irradiation), a triple staining was done with

antibodies against TH, k27ac and γH2AX is done. This research was done on experiment group 5 (table 7).

Table 7 experiment group 5, C57bl/6 mice are exposed to full body irradiation with different doses at 10 days of age. Mice were collected at 4 and 24 weeks after irradiation.

Age Doses of ionizing radiation

4 weeks - CTRL - 0.1Gy - 0.5Gy - 2Gy 24 weeks - CTRL - 0.1Gy - 0.5Gy - 2Gy

The protocol in appendix 1 is used with the specific content presented in table 8. Table 8 Specific content IF triple staining TH-K27ac- γH2AX

Content

2nd_{primary AB} _{K27ac (rabbit, AB-4729, 1:1000)}

γH2AX(mouse, 05-636, 1:1000)

2nd_{secondary AB} _{Against K27ac: Alexa 647 (A31372, 1:500)}

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4.9 Detection of gene silencing in 4week and 24 week old mice, exposed to

ionizing radiation

Ionizing radiation is said to have effect on transcription levels in DA neurons. To determine the effect of different doses of ionizing radiation on the gene silencing levels at young mice (4 week after irradiation) compared to adult mice (24 week after irradiation), a triple staining was done with antibodies against TH, 5-mc and γH2AX is done. This research was done on experiment group 5 (table 7). Again the protocol in appendix 1 was used with the specific content presented in table 9.

Table 9 Specific content IF triple staining TH-5mc-γH2AX Content

2nd_{primary AB} _{5-mc (mouse, Millipore, MABE146,1:1000)}

γH2AX (rabbit, ab2893, 1:1000)

2nd_{secondary AB} _{Against 5-mc: Alexa488 (A21202, 1:500)}

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5. Results

5.1 BrdU pilot staining – trial #1

We performed different trials for the optimization of the BrdU incorporation. In figure 9 a schematic scheme shows the experimental design of the different trials.

Figure 9 A schematic scheme explaining different trials. Approximately 10 day old c57bl6 mice are used in this experiment. BrdU injection was done, followed by immediate irradiation with 2Gy. In trial #1 and #2 there was waited for 2 hours for incorporation of BrdU, sacrificing of mice was done after 2 hours. In trial #3, mice were sacrificed after 2, 4 and 6 hours of BrdU incorporation.

5.1.1 Detection of neurogenic cells in the subventricular zone

Figure 10 Laser microscopy images of BrdU pilot staining. C57Bl6 mice were pre-treated with 0.2mg/g BrdU. Detection of BrdU was done by an anti-BrdU antibody (DAKO, 1:100), combined with a cy3 secondary antibody. Staining was performed on sections containing subventricular zone. Figure A,B and C represent group displayed in table 1. It is clearly to see that the images give promising results and BrdU is detected.

Before using BrdU to monitor DNA repair capacity, substantial pilot experiments were needed to define the optimal technical and practical conditions to use BrdU incorporation in brain tissue. Figure 10 shows results obtained by using laser microscopy on sections containing the subventricular zone, which is one of the principal sites of brain neurogenesis, along with the Dentate gyrus. Results were acquired after using the first pilot protocol for BrdU staining, as described in chapter 4.1 and 4.1.2. Figure A, B, and C represent the groups displayed in table 1. The results from either group A, B, or C shows succesful

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incorporated and detection of BrdU in newly formed neurons in the SVZ.

5.1.2 HCl treatment decreases effect TH staining

Efficient detection of BrdU requires an antigen retrieval step in acidic environment. Initially, we wanted to assess whether this process was compatible with the other staining used in our setting, the TH one. The antigen penetration improvement step is done by incubation in 0.1M HCl, followed by incubation in 2M HCl. The HCl will create a highly acidic environment. In the very first pilot experiments, we could not detect any TH signal after antigen retrieval. We therefore sought to optimize also these conditions to obtain clear TH signal after HCl treatment. A staining with the groups showed in table 2 was done to determine if the HCl treatment decreased TH functioning and revealed that HCl treatment indeed decreases sensitivity of TH staining.

Figure 11 Confocal microscope images (63x) showing HCl sensivity to TH staining using TH antibody dilution of 1:4000. Left: TH staining using normal IHC protocol, no HCl treatment. The image shows TH staining worked, however not optimal. Right: TH staining using IHC protocol with HCl treatment, antibody dilution 1:4000. The staining of DA neurons in with this protocol is barely visible.

This is exemplified in the images displayed in figure 11 showing the results of a TH staining with an antibody concentration of 1:4000 with (right image) or without (left image) HCL treatment. Both images show low staining of TH-positive cells when using antibody concentration of 1:4000, but the signal is particualry low on the HCl treated sample (right). When not using the HCl treatment, the TH staining is has a sub-optimal sensitivity because only a few DA neurons are revealed clearly.

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Figure 12 Confocal microscope images (63x) showing HCl sensivity to TH staining using TH antibody dilution of 1:2000. Left: TH staining using normal IHC protocol, no HCl treatment. The image shows good results which could be used for further research. Right: TH staining using IHC protocol with HCl treatment. The DA neurons are better visible with the lower dilution of the TH antibody, however the staining did not work optimal under these conditions.

Figure 12 shows results from a TH staining with a TH antibody concentration of 1:2000. The left section stained without HCl treatment shows good results on which further research could be based on. Again the section stained with the HCl treatment, shows significantly lower TH staining.

In summary, incubation of the sections in a highly acidic environment containing HCl sharply decreases detection of DA neurons using the anti-TH antibody. Even when increasing the antibody concentrations, DA neurons were revealed with a lower sensitivity than in untreated controls. Due to the fact that the HCl treatment is needed for properly functioning of BrdU detection, a TH antibody concentration of 1:500 was used for the following staining.

5.2 BrdU pilot staining – trial #2

5.2.1 No DNA repair detected in DA neurons after short incorporation of BrdU

To monitor DNA repair capacity of DA neurons when exposed to ionizing radiation, a triple staining was done with antibodies for TH, BrdU, and 53BP1. The TH antibody will detect DA neurons, 53BP1 is a marker for DNA damage, and BrdU incorporates during the S-phase of the cell cycle of proliferating or repairing cells. The different groups used for this experiment are shown in table 1 and a schematic design of this experiment is shown in figure 9 (trial #2). In this experiment, BrdU has a relatively short incorporation time of 2 hours after exposure to ionizing radiation.

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Figure 13 Confocal microscope images of triple staining using antibodies for TH (red), BrdU (green) and 53BP1 (blue). Images show results from staining on the control group (C, table 1). N1= mouse 1, N2 = mouse 2. Left: 40x magnification images of DA neurons in substantia nigra. Right: 40x magnification images, zoomed in. DA neurons are clearly visible in both mice. DNA damage marker 53BP1 is barely detected in DA neurons. No BrdU incorporation revealed in both mice.

Figure 13 shows confocal images of the triple staining on the substantia nigra; the results are from group C (table 1), which represents the control group. The DA neurons are clearly revealed by the TH-positive cytoplasm shown in red. As expected the 53BP1 DNA damage marker showed almost no presence in DA neurons or other cells, since group C is the control group that has not been exposed to radiation. In both mice, no DNA synthesis was revealed by BrdU-positive labeling. These results were expected for the control group, since the DA neurons were neither replicating, nor damaged by radiation and thus activation of repair or replicating mechanisms is not expected.

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Figure 14 Confocal microscope images of triple staining using antibodies for TH (red), BrdU (green) and 53BP1 (blue). Images show results of staining on group A (table1). Left: 40x magnification images of DA neurons in substantia nigra. N1= mouse 1, N2 = mouse 2 Right: 40x magnification images, zoomed in. DA neurons are clearly visible. Because the mice were irradiated with 2Gy, DNA damage occurred. The DNA damage is revealed by 53BP1 (blue) and shows DNA damage in most of the TH stained DA neurons. In N1, BrdU signal is pointed out by the arrows. No co-localization between TH-BrdU-53BP1 was seen. N2 barely shows any staining of BrdU near DA neurons.

Figure 14 shows confocal images of the triple staining in the substantia nigra; the results are from group A (table 1), which represents a group exposed to 2Gy of ionizing radiation, 2 hours after being pre-treated with BrdU (0.2 mg/g). DA neurons are again clearly shown by TH-positive (red) cytoplasms. Because of exposure to 2Gy of ionizing radiation, almost all DA neurons and other surrounding cells are labeled with DNA damage marker 53BP1 (blue). BrdU labelled nuclei are detected by IHC, revealed by the green oval shapes. In one animal (mouse identification N1), the BrdU-positive nuclei are not found within the DA neurons cytoplasms, but only in other cells in their proximity (shown by arrows). In the other (mouse identification N2) there are some vaguely BrdU-positive cells seen in the vicinity of the DA neurons; further away BrdU-positive nuclei are present. The cellular phenotype of the BrdU-positive cells in the vicinity of the DA neurons cannot be determined unequivocally, but due to the location they are assumed to be satellite glial cells. No co-localization between TH, 53BP1 and BrdU was seen in group A. Co-localization between 53BP1 and BrdU was not present either, which assumes that the BrdU-positive satellite cells is not related to ongoing DNA repair.

Transcription and DNA damage dynamics in the dopaminergic system following low dose ionizing radiation.