In der vorliegenden Arbeit wurden einerseits zelltypspezifische Untersuchungen der mitochondrialen DNA zur Bestimmung der Deletionslast, als Marker für oxidativen Stress, andererseits neuroinflammations-assoziierte Genexpressions-Analysen am humanen post mortem Hirngewebe von Patienten mit unterschiedlichen Stadien der Alzheimer Erkrankung durchgeführt. Als Grundlage hierzu diente das noch nicht gänzlich aufgeschlüsselte Konzept der selektiven Vulnerabilität unterschiedlicher Hirnregionen. Dabei zeigte sich, dass der Hippocampus, eine auf lichtmikroskopischer Ebene sehr früh befallene Region, auch molekularbiologisch deutliche Unterschiede gegenüber resistenten Regionen wie z.B. dem Kleinhirn aufweist. / In the present study, cell-type specific mitochondrial DNA deletion levels, as marker for oxidative stress on the one hand and neuroinflammation-related gene expression on the other hand were analyzed on human post mortem brain tissue from patients with Alzheimer’s disease at different stages. The study is based on the concept of selective vulnerability of different brain regions, which is not yet fully understood. Microscopical early affected regions such as the hippocampus show differences also on molecular level compared to more resistant regions such as the cerebellum.

ABSTRACT Alzheimer's disease (AD) is the most common progressive age related dementia and the fourth major cause of mortality in the elderly in the United States. AD is pathologically characterized by deposition of amyloid beta (Aβ) plaques in the brain parenchyma and neurofibrillary tangles (NFTs) within the neuronal soma. While pharmacological targets have been discovered, current strategies for the symptomatic or disease-modifying treatment of AD do not significantly slow or halt the underlying pathological progression of the disease. Consequently, more effective treatment is needed. One possibility for amelioration is using human umbilical cord blood cell (HUCBC) therapy. HUCBCs comprise a population of hematopoietic stem and progenitor cells. During recent years, functional recovery has been observed from the use of HUCBCs in pre-clinical animal models of brain and spinal cord injuries. Thus, modulation by cell therapy, specifically HUCBCs, may be a suitable treatment for AD and other models because of the observed cognitive and behavioral improvements. The studies presented in this dissertation centers on the suitability of using HUBCs as a potential treatment for AD. Expanding on this, the aims of the study sought to: (I) Investigate bio-distribution of HUCBC transplantation in PSAPP mice, (II) Characterize efficacy and determine therapeutic outcome of HUCBC following short and long term multi injections at early and late disease stages in PSAPP mice and (III) Determine AD-like pathological and cognitive changes associated with multiple HUCBC-derived monocyte (CD14) injections in PSAPP mice. Thus the findings of this work evolved from experiments that characterized the effects of low-dose infusions of HUCBC and HUCBC-derived monocytes into 6 month old Presenilin 1/Amyloid Precursor Protein (PSAPP) plaque-developing transgenic AD mice. Treated mice were studied using standard behavioral tests to determine the effects of infusion on the multiple cognitive domains affected by AD, followed by biochemical and histological analyses that included Aβ load and amyloid precursor protein (APP) processing. Specifically, PSAPP mice and their wild-type (WT) littermates were treated monthly with a peripheral HUCBC infusion over a period of 6 and 10 months, followed by cognitive and motor evaluation. Additionally, based on reports that tumor cells can originate from stem cells present in HUCB, we further examined whether monocytes purified from HUCBCs would have a similar significant effect on the reduction of AD-like pathology in PSAPP mice. HUCB cells homed into tissues including the brain. The principal finding was significant reduction in Aβ levels and β–amyloid plaques following low-dose infusions of both HUCBC– derived mononuclear cells as well as HUCBC-derived monocytes, with the monocytes providing a stronger effect. Results further demonstrated that HUCBC and HUCBC– derived monocyte infusion could improve memory function and locomotor ability in treated PSAPP mice. A possible reason for behavioral improvements in these animals may be the significant reduction in both Aβ levels and plaque load. This study also identified significant reduction in microglial activation and astrocytosis, both of which can contribute to AD pathology. In conclusion, our data suggest that it might be the HUCBC–derived monocytic population rather than stem cells that are responsible for the reduction in AD pathology.

L’activació microglial és un fenomen fisiològic que permet resoldre alteracions a l’homeostasi. Quan l’estímul desencadenant de l’activació microglial és proinflamatori, es desencadena una resposta inflamatòria en la que la micròglia produeix citocines i altres factors proinflamatoris que tenen potencial neurotòxic. Si aquesta activació es dóna de forma exacerbada o es cronifica, pot acabar tenint un efecte perniciós. De fet, diversos autors postulen que l’activació microglial pot agreujar el curs de patologies com l’Alzheimer o el Parkinson. És per això que en aquesta tesi ens vam plantejar la inhibició del fenotip proinflamatori de l’activació microglial com una diana terapèutica en les malalties neurodegeneratives. El primer objectiu va ser establir i caracteritzar un model in vitro de neuroinflamació, en el que estudiar el patró d’activació microglial a cultius tractats amb un estímul proinflamatori (LPS + interferó gamma). Així, vam determinar que la micròglia estimulada amb LPS/IFN-gamma s’activa amb un patró de resposta proinflamatori que es caracteritza per un increment en l’expressió dels enzims iNOS i COX-2 i en la producció de NO, TNF-alfa i IL-6. En una segona fase es va establir i caracteritzar un model de cocultiu de neurones i micròglia on estudiar la neurotoxicitat induïda per activació microglial en el que vam determinar que l’activació de la micròglia en resposta al LPS/IFN-gamma té un efecte neurotòxic principalment mediat per la producció de NO, mentre que el TNF-alfa la IL-6 i la COX-2 tenen un paper secundari. Una vegada establert el model experimental vam abordar la modulació de l’activació microglial inhibint els factors implicats en l’activació proinflamatòria, en concret els C/EBPs, ja que aquests factors regulen l’expressió de molts dels gens implicats en la resposta proinflamatòria microglial. La nostra hipòtesi era que la inhibició dels C/EBPs disminuiria aquesta resposta i tindria un efecte neuroprotector. La inhibició es va abordar des d’una aproximació farmacològica i una aproximació genètica. La inhibició farmacològica es va fer usant el flavonoide crisina que té efectes antiinflamatoris mediats per la inhibició dels C/EBPs en macròfags. Així, vam determinar que la crisina té un efecte antiinflamatori i neuroprotector i que aquest està mediat, si més no en part, per la inhibició de C/EBP-delta. Posteriorment, mitjançant la utilització de micròglia de ratolins deficients en C/EBP-beta o C/EBP-delta, vam determinar que l’absència de C/EBP-beta o C/EBP-delta inhibeix l’activació microglial i la neurotoxicitat associada. Els C/EBPs per tant són una possible diana terapèutica addicional per al tractament de patologies que cursen amb neuroinflamació. Per últim es va plantejar la inhibició de la sobreactivació microglial proinflamatòria potenciant els senyals inhibidors que mantenen a la micròglia en un estat quiescent, en concret la parella lligand-receptor CD200-CD200R. La nostra hipòtesi era que modulant l’activació de CD200R es podria inhibir la sobreactivació microglial i la neurotoxicitat associada. Així el tercer objectiu va ser estudiar, d’una banda quins estímuls poden modular l’expressió de CD200 a les neurones, i de l’altra quin és el paper de CD200 i CD200R en la modulació de l’activació microglial per part de les neurones. Per abordar aquest objectiu es va establir un model de mort neuronal apoptòtica i un model de mort neuronal necròtica per excitotoxicitat. En aquest model vam determinar que l’expressió de CD200 augmenta en neurones que estan morint, ja sigui per apoptosi com per necrosi, el que podria constituir un mecanisme de control de l’activació microglial en presència de mort neuronal (no es pot descartar que l’efecte observat sigui degut a una major estabilitat CD200 en front a altres proteïnes neuronals). Per últim ens vam plantejar estudiar l’efecte de la modulació de la senyalització CD200-CD200R sobre l’activació microglial i la neurotoxicitat associada, demostrant quel’expressió proteica de CD200 a neurones no es modula per estímuls antiinflamatoris com la IL-4 i la IL-10 ni proinflamatoris com el LPS i l’IFN-gamma. En resum, els resultats d’aquesta tesi mostren la utilitat dels models experimentals in vitro per estudiar la resposta inflamatòria de la micròglia i la neurotoxicitat resultant. Amb aquests models hem pogut establir la importància de la família de factors de transcripció C/EBPs en la regulació de la resposta inflamatòria microglial i suggerir la rellevància de CD200 en la comunicació entre la micròglia i les neurones que han entrat en un procés de mort cel•lular. / Microglial activation is a physiologic phenomenon that occurs in response to alterations of homeostasis. When microglia are activated by an pro-inflammatory stimulus there is an inflammatory response where cytokines and other pro-inflammatory factors with neurotoxic potential are released. A chronic or exacerbated response can have harmful effects; in fact many authors connect microglial pro-inflammatory overactivation with degenerative diseases such as Alzheimer’s or Parkinson’s disease. Given that, the main goal of this thesis was develope strategies to inhibit pro-inflammatory microglial activation that could serve as therapeutic target in neurodegenerative diseases. To do so we set up and characterized an in-vitro model of neuroinflammation using LPS + interferon-gamma as a proinflammatory stimulus. Treated microglial cells undergo a proinflammatory response pattern characterized by the release of NO, TNF-alpha and IL-6 and the increase of iNOS and COX-2 expression. When microglial cells are co-cultured with cortical neurons, the pro-inflammatory response results in neurotoxicity, an effect mediated mainly by NO release, whereas TNF-alpha, la IL-6 and COX-2 may have a secondary role in the process. Using this model we tested whether we could modulate microglial activation by inhibiting C/EBPs, a family of transcription factors involved in the regulation of pro-inflammatory genes. We first used a pharmacologic approach treating microglial cells with an anti-inflammatory compound named crisin. We determinated that crisin has anti-inflammatory and neuroprotective effects and that these effects are mediated,at least in part, by C/EBP-delta inhibition. We then used a genetic approach to inhibit C/EBPs, by using microglial cells from KO mice. We showed that the lack of C/EBP-beta or C/EBP-delta inhibits microglial activation and the associated neurotoxicity. Therefore, we suggest that C/EBPs are a potential therapeutic target in the treatment of diseases where neuroinflammation is present. We also modulated microglial activation by potentiating the inhibitory signals that keep microglia in a resting state, such as the neuronal ligand CD200 and its microglial receptor CD200R. We showed that CD200 expression appears to increase in response to neuronal death but not in response to pro-inflammatory stimuli such as IL-4, IL-10 or anti-inflammatory stimuli such as LPS and IFN-gamma. In summary we demonstrated that in-vitro models can be used to study the pro-inflammatory and neurotoxic effect of the microglial response. Using this approach we determinated the relevance of C/EBPs in regulating microglial activation and our studies support an important role of CD200 in the crosstalk between microglia and neurons that undergoing cell death.

A novel cell culture system was designed to serve as a model of neuroinflammation. Neurons, astrocytes, and microglia derived from embryonic and perinatal rat cortical tissue were combined in a three-dimensional hydrogel utilizing a method that facilitated cell maturation and viability. Chemical challenge of the cultures with a broad pro-inflammatory stimulus resulted in the production of inflammatory cytokines and other associated molecules commensurate with the response observed in vivo and in other in vitro systems. It was hypothesized that mechanical deformation of the multitypic neural cell cultures would produce a similar response and thus validate the system as an in vitro model of traumatic brain injury-induced neuroinflammation. Mechanical injury delivered using custom-manufactured culture chambers and injury devices successfully imparted a moderate level of cell death to the cultures. It was determined that a mechanically-induced inflammatory response required chemical stimulation prior to the injury. The research presented here describes the generation and characterization of a novel in vitro culture system and its implementation in experiments designed to model secondary injury mechanisms associated with injury-induced neuroinflammation. The findings of these studies, applications of the culture system, and future research avenues are discussed.

Intracerebral hemorrhage (ICH) is a devastating form of stroke that results from rupture of a blood vessel in the brain. Tissue inside the hematoma is irreversibly damaged soon after ICH onset and when this thesis research began, there was a dearth of information regarding pathological changes outside the hematoma. Inflammation is often proposed as a mechanism of injury, but very little information was available to show that inflammatory cells were in the right place at the right time to cause secondary brain injury. Using the collagenase-induced model of ICH, this work sought to better define spatial and temporal relationships between secondary brain injury and the inflammatory response after ICH. To test the hypothesis that reducing inflammation can protect the brain from secondary injury, minocycline, an antibiotic with established anti-inflammatory effects, was administered 6 hours after ICH onset. A small number of neurons die in the parenchyma bordering the hematoma between 6 hours and 3 days after ICH onset. This area was not associated with neutrophil infiltration, and most activated microglia/macrophages did not accumulate until after most neuron death had occurred. Despite a pronounced microglial response and prolonged increase in expression of many inflammatory genes, including complement receptor-3, interleukin-1 beta, interleukin-6, and interleukin-1 converting enzyme, no dying neurons were observed further outside the hematoma at any time. Interestingly, less early neuron death was observed in aged than in young animals, without a concomitant difference in the amount of tissue lost at 28 days. However, aged animals had less early microglial activation and a larger residual lesion, which might have resulted from impaired phagocytosis by activated microglia/macrophages. Minocycline was less effective in reducing microglial activation in aged animals, and did not reduce neuron death in either young or aged animals. Edema and BBB disruption was associated with degradation of the basal lamina protein, collagen type IV, and that damaged vessels are associated with tumor necrosis factor-alpha (TNFα)-positive neutrophils and active matrix metalloprotease-12 (MMP-12), all of which were reduced by delayed minocycline treatment. In contrast to ischemic stroke, there is a limited ‘penumbra’ outside the hematoma. Nevertheless, BBB damage in this region appears to be a potential target for protection. Furthermore, the prominent inflammatory response that continues for days after ICH does not appear to be associated with damage to other areas of the brain. Minocycline appears to protect the BBB by reducing neutrophil infiltration and the MMP-12 mediated basal lamina degradation. Future studies should investigate other targets for protection (i.e., white matter injury), and seek drugs that modulate the inflammatory response in aged animals and promote lesion resolution.

Microglia, the resident immune cells of the central nervous system, can become activated following injury, disease, or infection. In vitro, they can be activated by stimuli, which determine the inflammatory phenotype they will develop. In this thesis, stimulating microglia with tumor necrosis factor- and interferon- resulted in classical activation, characterized by proliferation, increased transcription of complement receptor 3 and major histocompatibility class II molecules, and elevated production and transcription of interleukin-1 and nitric oxide. Stimulation with TNF and IFN also changed the intensity of phosphorylated (activated) cyclic adenosine monophosphate response element binding protein immunoreactivity in microglia. Specifically, cells differentiated into populations with high or low pCREB intensity. This was the first example of such a response in microglia and was representative of what occurred in vivo, after ICH. Thus, the characterization of this model will be useful for future studies of this and other intracellular pathways of classically activated microglia.

Alzheimer’s disease (AD) is a multifactorial disease that results in progressive neurodegeneration. Brain regions are differentially affected in AD; some are more vulnerable to degeneration than others. There is an age-dependent effect on beta-amyloid (Aβ) accumulation and neuroinflammation as disease progresses. In the TgCRND8 APP transgenic mouse model, levels of various Aβ species and cytokines were determined as a function of brain region and age. Aβ was found to accumulate in the brain prior to the sequential elevation of IL-1β and CXCL1. Levels of Aβ, IL-1β and CXCL1 were elevated in regions that are severely affected in AD patients. It has been shown for the first time in an APP transgenic model that CXCL1 elevation occurs following IL-1β elevation.

Alzheimer’s disease (AD) is a multifactorial disease that results in progressive neurodegeneration. Brain regions are differentially affected in AD; some are more vulnerable to degeneration than others. There is an age-dependent effect on beta-amyloid (Aβ) accumulation and neuroinflammation as disease progresses. In the TgCRND8 APP transgenic mouse model, levels of various Aβ species and cytokines were determined as a function of brain region and age. Aβ was found to accumulate in the brain prior to the sequential elevation of IL-1β and CXCL1. Levels of Aβ, IL-1β and CXCL1 were elevated in regions that are severely affected in AD patients. It has been shown for the first time in an APP transgenic model that CXCL1 elevation occurs following IL-1β elevation.

Bien que le streptocoque de groupe B (SGB) soit une des bactéries les plus fréquentes au cours de la grossesse, l’impact de l’inflammation maternelle induite par le SGB sur le cerveau en développement n'a jamais été étudié. Selon notre hypothèse, l’activation immunitaire maternelle relevant de la présence du SGB en fin de grossesse joue un rôle dans la genèse des dommages cérébraux périnataux et des troubles neurodéveloppementaux qui en découlent, par exemple l’autisme. D'ailleurs, la génétique ne permet d'expliquer qu'environ 10 à 20% des cas d'autisme. De plus en plus d'études épidémiologiques tendent à montrer que les facteurs environnementaux sont aussi à considérer dans les origines de l’autisme. Nos résultats, issus d'un nouveau modèle animal pré-clinique d'inflammation maternelle induite chez le rat par le SGB, montrent des dommages à la fois placentaires et cérébraux ainsi que des troubles comportementaux dans la progéniture. Contrairement aux résultats obtenus à l’aide d'autres modèles d'inflammation prénatale induite par des bactéries Gram négatives (agissant principalement via le Toll-like receptor (TLR)-4) ou par des composants viraux (TLR-3), l’inflammation maternelle induite par la bactérie Gram positive (TLR-2) SGB entraîne un patron distinctif de chorioamnionite et de lésions cérébrales. Une dysmyélinisation sous-corticale est observée dans le prosencéphale mais toutefois, il n'y a aucune prolifération des cellules gliales. Des traits comportementaux associés à l’autisme, notamment des déficits de la communication et de la socialisation, ont été remarqués dans la progéniture exposée au SGB. Cette combinaison de traits associés à l’autisme est présente seulement chez les mâles. Nos résultats montrent pour la première fois que la réponse inflammatoire maternofoetale au SGB joue un rôle dans l’induction de lésions placentaires et cérébrales, et récapitule plusieurs caractéristiques cardinales de l’autisme chez l’humain telle que la susceptibilité liée au genre, la dysconnectivité cérébrale ainsi que plusieurs traits comportementaux typiques. Cela demeure un défi de modéliser les troubles du spectre autistique, cependant, ces résultats appuient les théories selon lesquelles, au-delà de l’influence génétique, les facteurs environnementaux jouent un rôle dans l’autisme et sa prévalence selon le genre.

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