Ageing is a process correlated with cellular stress and increased risks of neurodegenerative diseases, in particular Alzheimer’s disease (AD), which is accompanied with severe cognitive and memory impairments. Both ageing and AD affect many brain regions and thus induce brain malfunctions. Among the brain regions, the entorhinal cortex (EC) has drawn more and more attentions due to its pivotal role in cognition and memory functions as well as its vulnerability to ageing process and AD neuropathology. Synaptic and neuronal degenerations, which are also manifest features of AD, occur in the EC during the ageing process and at the early stage of AD. In addition, both pathological hallmarks of AD, namely abnormal accumulation of β-amyloid (Aβ) and hyperphosphorlation of tau proteins, initially appear in the EC and then progress to other brain regions such as the hippocampus and the neocortex. Glial alterations in AD and ageing process have been considered as secondary event to neuronal changes. Nevertheless, accumulating evidence indicates the relevant and primary involvement of astroglia, which is responsible for brain homeostasis, in AD and ageing. In this thesis, we have focused on the astroglial alterations in the EC during the progression of AD in an animal model of the disease as well as in ageing process in non-transgenic control mice. We have used the triple transgenic mouse model of AD (3xTg-AD), which is the most relevant animal model of AD and resembles the spatiotemporal progression of human AD pathology. Our results revealed cytoskeletal atrophy of astrocytes in the EC of 3xTg-AD animals (Chapter 3), shown by significant decrease in GFAP surface and volume. This astroglial alteration began at very early age (1 month) and sustained till more advanced age (12 month). Moreover, Aβ plaques did not trigger astrogliosis, and there was rare presence of GFAP labelled astrocytes in the vicinity of Aβ deposition. This may reflect the relative indifference of astroglia in the EC and thus explain the susceptibility of the EC at the early stage of AD. To study whether astroglial atrophy in cytoskeleton compromise astrocytic function in glutamate homeostasis, we investigated the expression of glutamine synthetase (GS), which is specifically expressed in astrocytes and is critical for glutamate balance (Chapter 4). Our results showed constant GS expression and the density of GS positive astrocytes in the EC. However, dual labelling of GS and GFAP revealed 3 different subsets of astrocytes, being GS-, GFAP-, GS/GFAP- positive astrocytes. The morphology of GS-IR cells, measured by surface and volume, did not change in spite of the evident GFAP atrophy. Therefore, GFAP atrophy does not disturb glutamate homeostasis in the EC, suggesting diverse functional populations of astrocytes, which may show distinct responses during AD progression. In addition we also analysed astroglial changes during the ageing process in the EC and its major projection area, the hippocampus (Chapter 5). Astrocytes in the hippocampus exhibited prominent hypertrophy, shown by increased GFAP whereas entorhinal astrocytes in the EC had profound reduction in GFAP expression. This may implicate heterogeneous astrocytic responses to ageing in different brain regions. The general atrophy of astrocytes in the EC of 3xTg-AD mice and aged controls, suggests astroglial atrophy may results in reduced astrocytic coverage and modulation of synapses, accounting for the synaptic dysfunction in ageing and AD.

Parkinson’s disease (PD) is a progressive neurodegenerative disease and characterized by the loss of dopaminergic (DA) neurons in the substantia nigra in the midbrain. The causes of the disease are still unknown. The most commonly used treatment is administration of L-DOPA, however, another possible treatment strategy is to transplant DA neurons to the striatum of PD patients to substitute the loss of neurons. Clinical trials have demonstrated beneficial effects from transplantation, but one obstacle with the grafting trials has been the variable outcome, where limited graft reinnervation of the host brain is one important issue to solve. To improve and control the graft DA nerve fiber outgrowth organotypic tissue cultures can be utilized. Cultures of fetal ventral mesencephalon (VM) have been used to investigate astrocytic migration and dopamine nerve fiber formations at different time points and under varying conditions to study how to control nerve fiber formation. The early appearing DA nerve fibers as revealed by tyrosine hydroxylase (TH) –immunoreactivity, form their fibers in the absence of glial cell bodies, are not persistent over time, and is called non-glial-associated TH-positive nerve fiber outgrowth. A monolayer of astrocytes guides a second persistent subpopulation of nerve fibers, the glial-associated TH-positive nerve fiber formation. Investigations of the interactions between the astrocytic migration and nerve fiber formations were made. In embryonic (E) day 14 VM cultures the mitosis of the astrocytes was inhibited with the antimitotic agent β-D-arabinofuranoside. The results revealed decreased astrocytic migration, reduced glial-associated TH-positive outgrowth, and enhanced presence of the non-glial-associated TH-positive outgrowth in the cultures. Thus, astrocytes affect both the non-glial- and the glial-associated growths by either its absence or presence, respectively. The astrocytes synthesize proteoglycans. Therefore the nerve fiber formation was studied in VM or spinal cord cultures treated with the proteoglycan blockers chondroitinase ABC (ChABC), which degrades the proteoglycans, or methyl-umbelliferyl-β-D-xyloside (β-xyloside), which blocks the proteoglycan synthesis. β-xyloside inhibited the migration of the astrocytes and the outgrowth of the glial-associated TH-positive nerve fibers in both VM and spinal cord cultures, whereas ChABC treatment had no effect in E14 VM or spinal cord cultures. E18 VM and spinal cord cultures were evaluated to investigate how the different developmental stages influence astrocytes and the two nerve fiber formations after 14 DIV. No nerve fiber formation was found in E18 VM cultures, while the non-glial-associated nerve fiber outgrowth was obvious as long and robust fibers in E18 spinal cord cultures. The astrocytic migration was similar in VM and spinal cord cultures. β-xyloside and ChABC did not affect nerve fiber growth but astrocytic migration in E18 VM cultures, while no effects was found in the spinal cord cultures. However, the neuronal migration found in control cultures was abolished in both VM and spinal cord cultures after both ChABC and β-xyloside. Neuroinflammation plays a critical role in the development of PD. Increased levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) are observed in postmortem PD brains and the levels of TNFα receptors on circulating T-lymphocytes in cerebrospinal fluid of PD patients are increased. The effects of TNFα were studied on E14 VM cultures. The outgrowth of the non-glial-associated TH-positive nerve fibers was inhibited while it stimulated astrocytic migration and glial-associated TH-positive nerve fiber outgrowth at an early treatment time point. Furthermore, blocking the endogenous levels of TNFα resulted in cell death of the TH-positive neurons. Furthermore, cultures of E14 mice with gene deletion for the protein CD47 were investigated. CD47 is expressed in all tissues and serves as a ligand for the signal regulatory protein (SIRP) α, which promotes e.g migration and synaptogenesis. CD47-/- cultures displayed massive and long non-glial-associated TH-positive nerve fiber outgrowth despite a normal astrocytic migration and the presence of glial-associated TH-positive nerve fiber outgrowth. For the first time, it was observed that the non-glial-guided TH-positive nerve fiber outgrowth did not degenerate after 14 DIV. Taken together, there is an interaction between astrocytes and TH-positive nerve fiber formations. Both nerve fiber formations seem to have their task during the development of the DA system.

The main aim of this thesis was to assess if early deprivation (ED) and glucocorticoid (GC) treatment exert long-term effects on the volume of the brain regions implicated in responses to stress, and if it associates with alterations in the distribution and structure of astroglia, which are known to support brain plasticity. This study also investigated the effects of prenatal dexamethasone (Dex) treatment on selected brain receptors, namely the oxytocin and 5-HT1A receptors, as they are implicated in the regulation of responses to stress. In addition, in vitro effects of Dex on neural stem cells were studied, in order to explore the drug effects on cell proliferation and differentiation, and on glial cell markers. Unbiased stereological estimation was employed to determine the regional brain volume, astroglial morphology and total cell count. Peripheral quantitative computed tomography (pQCT) technique was used to quantify total brain volume. Autoradiography technique was employed to visualise and analyse oxytocin and 5HT-1A serotonin receptor binding using selective radioactive ligands. The results of the present study demonstrate that both ED and prenatal Dex exposure leads to long-term effects on hippocampal remodelling with volume losses and impoverished astroglial morphology in the form of reduced primary process length. The observed deterioration in astroglial morphology adds further evidence that astrocytic changes contribute to hippocampal volume losses, a phenomenon that deserves more research in the context of effects of corticosteroid overload and stress-related pathologies. The present results also demonstrate that prenatal Dex induces long-term effects at the level of central neuroregulatory processes. Thus significant region- and sex-dependent reductions or increases in the oxytocin and 5-HT1A receptor binding were observed. The in vitro study has shown that Dex affects both proliferation and differentiation of GFAP positive NSCs with no toxic effects as such. Overall, both early postnatal or prenatal manipulations that increase levels of stress and/or glucocorticoids as the chemical mediators of stress, lead to a long-term maladaptive brain remodelling with losses in the hippocampal volume, impoverishment of hippocampal astroglial morphology and changes in the properties of central regulatory receptors in the brain areas involved in the reaction to stress.

Since neuroinflammation is present in patients with mild cognitive impairment (MCI) andAlzheimer's disease (AD) and since cholinesterase inhibitors increases the level ofacetylcholine, the aim was to investigate whether inflammatory markers of cholinoceptive cellsare affected in these patients. Near a biological hallmark of AD, amyloid plaque, activatedastrocytes and microglia can be found and higher levels of proinflammatory cytokines, i.e. IL-1β. To study the inflammatory response, proteins GFAP and S100B are used as CSF glialmarkers. IL-1β can bind to the membrane-bound IL-1 receptor or soluble sIL-1β-RII. When IL-1β binds to the soluble receptor instead of the membrane-bound receptor, no intracellular signalpropagation occur, thereby sIL-1βRII exerts an antagonistic effect and diminishedinflammatory responses. Thus a reduction in ratio of IL-1β to sIL-1RII levels may be indicativeof anti-inflammatory response. Available data on CSF GFAP, S100B, IL-1β and sIL-1β-RIIlevels in AD patients and MCI patients was used. MCI group were longitudinally followedafter start of treatment with a cholinesterase inhibitor (ChEI). AD group had data from baselineand after short-term treatment with a ChEI. The statistics application StatView was used toanalyse data. The activity of the cholinesterase enzymes, BuChE and AChE showed significantinhibition in the CSF of the MCI patients compared to baseline CSF GFAP level wassignificantly lower in MCI than AD patients at baseline. The levels of both GFAP and S100Bwere increased with time in MCI patients to comparable levels in the AD patients, indicative ofastroglial activation in MCI patients. However, the ratio of IL-1β to sIL-1RII showed alongitudinal reduction in the MCI patients after the treatment with the ChEIso that this ratiowas significantly higher in AD than in MCI patients. Thus despite the activation of astroglialcells in the treated MCI patients the proinflammatory effect of IL-1β was prevented byinduction of sIL-1βRII levels indicative of an anti-inflammatory outcome of treatment. Thisstudy suggests that proper activation of astroglial cells may have beneficial effect on ADpathogenesis, and conversion of MCI to AD. It also suggests that cholinesterase inhibitors may have an anti-inflammatory effect.

The discovery of a method by which proteins of interest can selectively be labeled with a probe of choice intracellularly is a longstanding goal in chemical biology research. Conventional labeling techniques have utilized large domain tags but despite the development of small labeling molecules there have been no short peptide sequences known to covalently label a small molecule without the aid of an enzymatic process or metal chelation. We aimed to find a sequence of nucleophilic peptides that reacted covalently and specifically with electrophilic small labeling molecules using mRNA display. Our goal was to show that an electrophilic small labeling molecule that is brought in proximal distance to a protein of interest via affinity can result in nucleophilic attack by a neighboring nucleophilic amino acid to covalently label the protein of interest. Utilizing affinity between a small labeling molecule and a protein of interest to bring them spatially close to one another maximizes the chance that a covalent reaction can take place and provides selectivity between two components in a complex mixture. Towards this goal, we developed several electrophilic fluorescent small molecules. Covalent labeling was achieved using electrophilic bait in the form of sulfonate esters, a polyethylene oxide linker provided structural flexibility, and a fluorescent affinity tag containing a rhodamine backbone served as the potential binding site to a key peptide sequence encoded within a protein of interest. The synthetic routes to access our electrophilic rhodamine B and sulforhodamine 101 fluorophores were optimized. Key intermediates were produced and served as flexible points of modification to make various analogs of our desired electrophilic fluorophores. The affinity between proteins containing the peptide sequence and the fluorescent electrophiles were determined by fluorescence polarization. Covalent labeling was determined to be both time and concentration dependent. The expected published affinity between the peptides and fluorophore was not high enough to produce selective labeling. However, our small labeling molecules were found to be effective at labeling various proteins in vitro. In addition, our electrophilic fluorophores have been found superior to sulforhodamine 101 in live cell imaging of astrocytes.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-08-25T20:54:16Z No. of bitstreams: 1 MalekChouchane_TESE.pdf: 3043835 bytes, checksum: b90ef34a2d4072ef5abda48d216aebb4 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-08-26T21:40:31Z (GMT) No. of bitstreams: 1 MalekChouchane_TESE.pdf: 3043835 bytes, checksum: b90ef34a2d4072ef5abda48d216aebb4 (MD5) / Made available in DSpace on 2016-08-26T21:40:31Z (GMT). No. of bitstreams: 1 MalekChouchane_TESE.pdf: 3043835 bytes, checksum: b90ef34a2d4072ef5abda48d216aebb4 (MD5) Previous issue date: 2016-02-29 / Recently, the field of cellular reprogramming has been revolutionized by works showing the potential to directly lineage-reprogram somatic cells into neurons upon overexpression of specific transcription factors. This technique offers a promising strategy to study the molecular mechanisms of neuronal specification, identify potential therapeutic targets for neurological diseases and eventually repair the central nervous system damaged by neurological conditions. Notably, studies with cortical astroglia revealed the high potential of these cells to reprogram into neurons using a single neuronal transcription factor. However, it remains unknown whether astroglia isolated from different regions of the central nervous system have the same neurogenic potential and generate induced neurons (iN) with similar phenotypes. Similarly, little is known about the fate that iNs could adopt after transplantation in the brain of host animals. In this study we compare the potential to reprogram astroglial cells isolated from the postnatal cerebral cortex and cerebellum into iNs both in vitro and in vivo using the proneural transcription factors Neurogenin-2 (Neurog2) and Achaete scute homolog-1 (Ascl1). Our results indicate cerebellar astroglia can be reprogrammed into induced neurons (iNs) with similar efficiencies to cerebral cortex astroglia. Notably however, while iNs in vitro adopt fates reminiscent of cortical or cerebellar neurons depending on the astroglial population used for reprogramming, in situ, after transplantation in the postnatal and adult mouse brain, iNs adopt fates compatible with the region of integration. Thus, our data suggest that the origin of the astroglial population used for lineage-reprogramming affects the fate of iNs in vitro, but this imprinting can be overridden by environmental cues after grafting.

Die Glutaminylzyklase QC und ihr Isoenzym isoQC katalysieren die Umwandlung N-terminaler Glutaminylreste an einer Reihe von Neuropeptiden, Peptidhormonen und Chemokinen in Pyroglutamat. Diese post-translationale Modifikation schützt Proteine vor proteolytischem Abbau und trägt auch zu ihrer biologischen Aktivität bei. Im Falle der Alzheimerschen Erkrankung führt QC/isoQC-Aktivität allerdings zur Stabilisierung des Aβ-Peptids und begünstigt damit dessen Akkumulation und Aggregation in Amyloid-Plaques. Beide Enzyme unterscheiden sich in ihrer zelltypspezifischen Expression und ihrer Substratspezifität in vivo. Die QC kommt eher in neuronalen Geweben als sekretorisches Enzym vor, während die isoQC ubiquitär exprimiert wird und im Golgi-Apparat verankert ist. Im Gegensatz zur QC ist die isoQC beispielsweise in der Lage, das proinflammatorische monocyte chemoattractant protein 1 (MCP1, auch CCL2 genannt) in seinen aktiven Zustand zu überführen. In der vorliegenden Arbeit sollte deshalb untersucht werden, inwieweit beide Isoenzyme in chronische Entzündungsprozesse involviert sind. Als Modell für diese Untersuchungen wurde die systemische Cuprizon-Applikation als tierexperimenteller Ansatz für die nicht-invasive Induktion einer Entzündungsreaktion im Gehirn gewählt, um folgende Fragestellungen zu beantworten: 1. Lässt sich die Entzündungsreaktion, die durch Cuprizongabe ausgelöst wird, mittels Iba1-, GFAP- und Hitzeschockprotein 27 (Hsp27)-Immunhistochemie nachweisen? 2. Sind QC und isoQC an durch Cuprizon ausgelösten entzündlichen Prozessen beteiligt? 3. Unterscheiden sich QC- und isoQC-knock-out (ko)-Mäuse hinsichtlich der Entzündungsreaktion unter Cuprizongabe? Dazu wurden Wildtyp (WT)-Mäuse (n = 6), QC-ko (n = 3) und isoQC-ko (n = 3) Mäuse einer sechswöchigen Gabe von 0,3 % Cuprizon im Futter unterzogen, was Tagesdosierungen von 12,9–13,5 mg Cuprizon pro Tier entspricht. Das Cuprizon als Kupferchelator löst einen Demyelinisierungsprozess ähnlich der histopathologischen Veränderungen bei Multipler Sklerose aus, der mit Chemotaxis von Immunzellen und Aktivierung von Gliazellen einhergeht. Als Kontrollgruppe wurden je vier unbehandelte Geschwistertiere der Genotypen WT, QC- und isoQC-ko-Mäuse herangezogen. In der Arbeit wurden folgende Ergebnisse erzielt: 1. Die Gliazellaktivierung wurde mit einer immunhistochemischen Färbung von Iba-1, einem Marker für Mikroglia, saurem Gliafaserprotein (GFAP) als Astrozytenmarker und dem Nachweis von Hitzeschockprotein 27 untersucht. Die Mikroglia- und Astrozytenaktivierung wurde durch Ermittlung des prozentualen Gliaanteils pro Fläche der jeweiligen Hirnregion erfasst, Hsp27-exprimierende Gliazellen wurden in jeder untersuchten Hirnregion an standardisierten mikroskopischen Aufnahmen des BZ-9000 Mikroskops der Firma Keyence unter Zuhilfenahme der BZ-II-Analyzer-Software manuell ausgezählt. Die ermittelten Flächen und Zellzahlen wurden mit der Graph-Pad-Prism 6-Software mittels ungepaarter t-Tests statistisch ausgewertet. Zu den Hirnstrukturen, die auf eine Entzündungsreaktion hin untersucht wurden, gehören das Corpus callosum, das Cingulum, die externe Kapsel, das Septum, das Striatum und dessen Faserbündel, der Cortex, die anteriore Kommissur, das ventrale Pallidum sowie der Hippocampus. Sowohl in Iba-1-, GFAP- und Hsp27-Färbung konnte bei den WT-Tieren in nahezu allen untersuchten Hirnregionen eine signifikante Erhöhung des Mikro- und Astroglia-Gewebeanteils sowie der Hsp27-Expression in Astrozyten nach Cuprizongabe gezeigt werden. Mikro- und Astrogliaaktivierung wurden bereits in früheren Studien mit Cuprizon-Applikation nachgewiesen, die Übereinstimmung der Ergebnisse zeigt die Wirksamkeit der Cuprizongabe und verdeutlicht die Entzündungsantwort auf zellulärer Ebene. Die Hsp27-Expression in Astrozyten konnte in dieser Arbeit erstmalig im Cuprizonmodell nachgewiesen werden und stellt eine neue, zelluläre Komponente der Entzündungsreaktion dar. 2. Mit Nachweis des Einflusses der isoQC auf die Aktivierung von CCL2 in peripheren Entzündungsmodellen vermutete man eine Beteiligung der Glutaminylzyklasen auch bei entzündlichen Prozessen im Gehirn. Es zeigte sich, dass QC-ko- und isoQC-ko-Tiere ohne Cuprizongabe im Mittel höhere gliäre Gewebeanteile und Hsp27-Expression aufwiesen als die unbehandelten WT-Geschwistertiere. In der hier stichprobenartig untersuchten Kohorte deutet dies auf eine Funktion der Glutaminylzyclasen für die Aktivierung von Gliazellen und eine teilweise Grundaktivierung in nativen QC-/ isoQC-ko-Tieren hin. Auf die Cuprizonbehandlung reagierten die ko-Tiere weniger stark als WT-Tiere mit Steigerung der Mikro- und Astrogliaaktivität oder erhöhter Hsp27-Expression. Das Fehlen der Glutaminylzyclasen führt somit möglicherweise zur Dämpfung einer chronischen Entzündungsantwort. 3. Inwiefern beide Glutaminylzyklasen zur vermuteten Entzündungshemmung beitragen, war ein weiterer Teil der Promotionsarbeit. Betrachtete man die Mikroglia in unbehandelten Tieren, zeigte sich in isoQC-ko-Mäusen eine höhere Grundaktivität als in WT- und QC-ko-Genotyp. Die dadurch im Verhältnis geringere Steigerung der Mikrogliaaktivität nach Cuprizongabe bei den isoQC-ko-Tieren im Vergleich zu Tieren des WT oder QC-ko-Genotyps könnte auf die fehlende Aktivierung des CCL2 zurückgeführt werden. Der isoQC-ko-Genotyp zeigte auch bei der Hsp27-Färbung unter den drei untersuchten Genotypen die geringste Steigerung der Hsp27-Expression nach Cuprizongabe, sodass die isoQC möglicherweise bei der Stimulation zur Expression dieses Hitzeschockproteins beteiligt ist. In den Iba-1-Färbungen zeigte sich auch, dass bei den QC-ko-Tieren unter Cuprizonapplikation Mikroglia in geringerem Maße aktiviert wird als bei WT- und isoQC-ko-Mäusen. Außerdem ergab die Auswertung der Hsp27-Expression von Astrozyten eine im Vergleich stark erhöhte Hsp27-Expression vor Cuprizongabe bei den QC-ko-Tieren. Das Fehlen der vorwiegend neuronal vorkommenden QC in den hier untersuchten zentralnervösen Strukturen hat möglicherweise gravierendere Auswirkungen auf zelluläre entzündliche Prozesse als das Fehlen der isoQC, die, ubiquitär vorkommend, weniger spezifische Aufgaben in Neuronen innehaben könnte. Die Arbeit zeigt, dass sich das Cuprizon-Modell eignet, einen chronischen Entzündungsprozess im Gehirn nachzustellen, um nachgeschaltete inflammatorische zelluläre Reaktionen eines Organismus zu untersuchen. Die Betrachtung von Mikrogliaaktivierung und Hsp27-Expression scheint für die Untersuchung des Cuprizon-Modells besser geeignet zu sein als die Auswertung der Astrogliaaktivität, da die fein nuancierten Unterschiede zwischen den betrachteten Glutaminylzyklasen in den GFAP-Färbungen weniger gut nachweisbar waren. Um die oben dargelegten Ergebnisse besser statistisch untermauern zu können, sollte in fortführenden Untersuchungen eine höhere Anzahl an Tieren pro Versuchsgruppe angestrebt werden.:Inhaltsverzeichnis Inhaltsverzeichnis I Abkürzungsverzeichnis III Abbildungsverzeichnis V Tabellenverzeichnis VII 1 Einleitung 1 1.1 Glutaminylzyklasen 1 1.2 Das Cuprizon-Modell 3 1.2.1 Einführung in das Demyelinisierungsmodell 3 1.2.2 Aktivierung von Gliazellen 4 1.3 Mechanismen von Entzündungsreaktionen 5 1.3.1 Mikroglia 6 1.3.2 Astroglia 7 1.3.3 Hsp27 8 1.4 Zielstellung 9 2 Material und Methoden 10 2.1 Vorgehen bei der Cuprizonbehandlung 10 2.2 Versuchstiere 10 2.3 Präparation und Aufarbeitung des Hirngewebes 11 2.4 Auswahl der Schnittebenen 12 2.5 Durchführung der immunhistochemischen Färbungen 16 2.6 Durchführung der Fluoreszenz-Färbungen 19 2.7 Geräte 20 2.8 Hard- und Software 20 2.9 Auswahl von Färbungen und Hirnregionen für die Auswertung 21 2.9.1 Auswahl und Auswertung der Iba1-Färbung 21 2.9.2 Auswahl und Auswertung der GFAP-Färbung 23 2.9.3 Auswahl und Auswertung der Hsp27-Färbung 24 3 Ergebnisse 25 3.1 Nachweis der Mikroglia-Aktivierung mittels Iba1-Färbung 26 3.1.1 Vergleich der unbehandelten Genotypen hinsichtlich des Gewebeanteils von Mikroglia 30 3.1.2 Einfluss der Cuprizonbehandlung auf die Mikrogliaaktivierung bei Wildtyp-Mäusen 33 3.1.3 Einfluss der Cuprizonbehandlung auf die Mikrogliaaktivierung bei QC-ko-Mäusen 35 3.1.4 Einfluss der Cuprizonbehandlung auf die Mikrogliaaktivierung bei isoQC-ko-Mäusen 36 3.1.5 Vergleich der behandelten Genotypen hinsichtlich der Mikrogliaaktivität nach Cuprizonbehandlung 38 3.2 Nachweis der Astroglia-Aktivierung mittels GFAP-Färbung 40 3.2.1 Vergleich der unbehandelten Genotypen hinsichtlich des Gewebeanteils von Astroglia 44 3.2.2 Einfluss der Cuprizonbehandlung auf die Astrogliaaktivierung bei Wildtyp-Mäusen 46 3.2.3 Einfluss der Cuprizonbehandlung auf die Astrogliaaktivierung bei QC-ko-Mäusen 48 3.2.4 Einfluss der Cuprizonbehandlung auf die Astrogliaaktivierung bei isoQC-ko-Mäusen 49 3.2.5 Vergleich der behandelten Genotypen hinsichtlich der Astrogliaaktivität nach Cuprizonbehandlung 50 3.3 Nachweis von Hsp27-Expression nach Cuprizonbehandlung 51 3.3.1 Vergleich der unbehandelten Genotypen hinsichtlich des Gewebeanteils von Hsp27-exprimierenden Astrozyten 53 3.3.2 Einfluss der Cuprizonbehandlung auf die Hsp27-Expression in Astrozyten bei Wildtyp-Mäusen 56 3.3.3 Einfluss der Cuprizonbehandlung auf die Hsp27-Expression in Astrozyten bei QC-ko-Mäusen 57 3.3.4 Einfluss der Cuprizonbehandlung auf die Hsp27-Expression in Astrozyten bei isoQC-ko-Mäusen 58 3.3.5 Vergleich der behandelten Genotypen hinsichtlich der Hsp27-Expression in Astrozyten nach Cuprizonbehandlung 59 4 Diskussion 61 Verweise X Zusammenfassung der Arbeit XXII Persönlicher und wissenschaftlicher Werdegang XXIV Eigenständigkeitserklärung XXV Danksagung XXVII

Die vorliegende Arbeit untersucht das Differenzierungsverhalten adulter muriner Knochenmarkzellen im Zentralnervensystem in vivo und in vitro. Hierzu wurden letal bestrahlte Mäuse mit Knochenmark aus transgenen Mausmutanten transplantiert, die das grün fluoreszierende Protein (GFP) unter der Kontrolle des humanen GFAP-Promoters exprimieren. Ein Teil der Rezipienten wurde vier Wochen nach Transplantation einer transienten fokalen cerebralen Ischämie unterzogen, um den Einfluss postischämischer inflammatorischer Vorgänge auf das Differenzierungsverhalten eingewanderter Zellen zu untersuchen. Eine zelluläre Koexpression von GFP und GFAP als Zeichen der Differenzierung hämatogener Zellen zu GFAP-exprimierenden Astrozyten fand sich bei keinem der analysierten Tiere. Für die in vitroVersuche wurden murine Knochenmarkzellen auf Mausastrozyten und auf organotypischen entorhinal-hippocampalen Hirnschnitten kokultiviert. Die hierzu verwendeten Knochenmarkzellen waren entweder retroviral mit GFP transfiziert oder stammten aus zwei verschiedenen transgenen Mausmutanten, von denen eine GFP nahezu ubiquitär unter dem b-Actin-Promoter, die andere GFP unter der Kon-trolle des humanen GFAP-Promoters exprimiert. Während zahlreiche Knochenmarkzellen nach wenigen Tagen der Kokultur die morphologischen Charakteristika ruhender Mikroglia annahmen und Immunoreaktivität für den Makrophagen/Mikroglia-Marker Iba1 aufwiesen, fand sich keine einzige Zelle mit Koexpression von GFP und GFAP. Diese Ergebnisse sprechen dafür, dass adulte murine Knochenmarkzellen bzw. ihre Abkömmlinge im zirkulierenden Blut nicht in GFAP-exprimierende Astrozyten differenzieren. / It has been postulated that adult murine bone marrow cells have the potential to differentiate into cells of neuroectodermal origin. In order to examine whether bone marrow cells can adopt an astroglial fate, various in vivo and in vitro approaches were chosen. Lethally irradiated recipient mice were transplanted with bone marrow derived from transgenic mice which express the green fluorescent protein (GFP) under the control of the human GFAP promoter. Four weeks after transplantation, several animals underwent transient focal cerebral ischemia. Although postischemic inflammatory processes may eventually have a permissive effect on cell differentiation, not a single cells coexpressing GFAP and GFP was found in the brains of all reci-pients examined. For in vitro studies, murine bone marrow cells were co-cultured on astrocytic monolayers or organotypic entorhinal-hippocampal brain slices. Bone marrow cells were either labelled by retroviral transfection with GFP or derived from two different transgenic mouse mutants expressing GFP under the control of the human GFAP-promoter or the murine b-Actin-promoter, respectively. After several days of co-culture bone marrow derived cells developed a ramified morphology and showed immunoreactivity for the monocytic/microglial marker Iba1. However, differentiation of bone marrow derived cells into GFAP-expressing astrocytes was not observed. Our results suggest that adult murine bone marrow cells cannot differentiate into GFAP-expressing astrocytes in vivo or in vitro.

Objective: Activated astroglia is involved in the pathophysiology of neurodegenerative diseases and has also been described in animal models of spinal muscular atrophy (SMA). Given the urgent need of biomarkers for treatment monitoring of new RNA-modifying and gene replacement therapies in SMA, we examined glial fibrillary acidic protein concentrations in cerebrospinal fluid (cGFAP) as a marker of astrogliosis in SMA. - Methods: 58 adult patients and 21 children with genetically confirmed 5q-associated SMA from four German motor neuron disease specialist care centers and 30 age- and sex-matched controls were prospectively included in this study. cGFAP was measured and correlated to motor performance and disease severity. Additionally, we compared fL). - Results: cGFAP concentrations did not differ from controls but showed higher levels in more severely affected patients after adjustment for patients’ age. Normalized cNfL values were associated with disease severity. Within 14 months of nusinersen treatment, cGFAP concentrations did not change, while cNfL decreased significantly. - Interpretation: cGFAP is not an outstanding biomarker in SMA, but might support the hypothesis that glial activation is involved in SMA pathology. Unlike previously suggested, cNfL may be a promising biomarker also in adult patients with SMA, which should be subject to further investigations.

No description available.