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NEURAL STEM CELL BIOLOGY AND NEUROGENESIS IN MOUSE MODELS OF AGING AND ALZHEIMER S DISEASE Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen
NEURAL STEM CELL BIOLOGY AND NEUROGENESIS IN MOUSE MODELS OF AGING AND ALZHEIMER S DISEASE Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Florian V. Ermini aus Basel (BS) Universität Basel 2006 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Markus Rüegg Prof. Dr. Mathias Jucker Dr. Matthias Staufenbiel Basel, den Prof. Dr. Hans-Jakob Wirz Dekan Table of Contents 1. Summary 1 2. Introduction Stem Cell Biology Background Adult stem cells Adult Mammalian Neurogenesis Neurogenesis in the adult brain Neurogenic regions The neurogenic niche Regulation of neurogenesis Neurogenesis and memory Neurogenesis in the aging hippocampus Alzheimer s Disease Clinical symptoms Pathophysiology Epidemiology and genetics of Alzheimer s disease APP processing and Aβ Mouse models of Alzheimer s disease Synthesis: Neurogenesis in the AD Brain Influence of APP and Aß on neurogenesis Angiogenesis in the AD brain Inflammatory factors and neurogenesis Loss of neurotransmitter affects neurogenesis Influence of presenilin on stem cell biology Aß induces sprouting on growing axons Hematopoietic stem cells in the AD brain References 26 3. Impact of age and caloric restriction on neurogenesis in the dentate gyrus of C57BL/6 mice Increased neurogenesis and alterations of neural stem cells in Alzheimer s disease mouse models Invasion of hematopoietic cells into the brain of amyloid precursor protein transgenic mice Conclusions 97 Abbrevations 100 Curriculum vitae 102 Bibliography 104 Acknowledgements I would like to specially acknowledge my supervisor Mathias Jucker for guiding me through the long path leading to a PhD, helping around some unexpected turns, while leaving me plenty of free choices to enjoy the journey. I would also like to thank Theo D. Palmer for introducing me to in vitro culturing of neural stem cells and enabling me to stay at his lab at Stanford University. Additionally, I would like to thank Matthias Staufenbiel for providing the APP23 mice and participating in the committee for this dissertation, Markus Rüegg for heading the dissertation committee, Donald Ingram for providing calorie restricted mice, Masohiro Yamaguchi for providing the nestin-gfp mice and Gerd Kermpermann for the helpful and open discussion of our results. Many thanks go to Luca Bondolfi and Anne Stalder for the collaborations that led to the publishing of some of the work presented in this thesis. The daily lab work is unbearable without good company, lunch pals and discussion partners, which I was very lucky to have plenty of by the members of the Jucker Lab at the Institute for Pathology in Basel and the Hertie Institute for Clinical Brain Research in Tübingen: Martin Herzig, Martina Stalder, Sonia Boncristiano, Stefan Käser, Janaky Coomaraswamy, Melanie Meyer-Lühmann, Michelle Pfeifer, Tristan Bolmont, Luca Bondolfi, Anne Stalder, Esther Kohler, Irene Neudorfer, Claudia Schäfer, Rebecca Radde, Bettina Braun, Michael Calhoun, Yvonne Eisele, Ana Fulgencio, Bernadette Graus, Ellen Kilger, Dennis Lindau, Jörg Odenthal and Lars Stoltze; and the members of the Palmer Lab at the Department for Neurosurgery at Stanford University: Klaus and Konstanze Fabel, Hiroki Toda, Akiko Mori, Michelle Monje, Robin Price and Eric Wexler. I d also like to thank Michael Calhoun for lots of scientific input and the occasional use of his futon. Thanks to the members Frisbee Club Freespeed Basel for helping me to keep my body and spirit in shape despite being tied to the computer, the microscope or the bench through these years. I would also like to express my gratitude and love to my parents Marco and Doris Ermini-Fünfschilling for the seemingly indefinite support they would never miss a chance to give. Thank you, Michelle Moore for all the joy, excitement and love we had because or despite this work! This work was supported by the Roche Research Foundation (Basel), Basler Stiftung für Demenzforschung (Basel) and the Swiss National Science Foundation. 1. Summary The etiology of Alzheimer s disease (AD) remains a great challenge for neurological research. Extensive investigations for almost one hundred years have led to profound insights of the pathological and molecular mechanisms that affect the AD brain, and there are several hypotheses about what causes the characteristic AD related dementia. The focus has fallen increasingly on the deposition of ß-amyloid (Aß) in the cortex and it is believed, that the generation and deposition of Aß is the leading cause of the disruptions observed in the AD brain. Aß has been shown to provoke neuron death, decreased synaptic plasticity, aberrant sprouting of growing axons, chronic inflammation and hyper-phosphorylation of tau. In recent years, research on adult neurogenesis in the mammalian brain has led to surprising findings: new neurons are added daily to specific regions of the brain and growing evidence suggests that these new neurons play a critical role for learning and memory, mood and, to a limited amount, repair of damaged cortical areas. All of these functionalities of neurogenesis are affected in AD patients and the question must be raised, if in the AD brain, neurogenesis is directly disturbed. Defects in neural stem cell biology might significantly contribute to AD dementia and the examination of the relationship of AD lesions and neural stem cell biology might provide new insights for the understanding and treatment of AD. Only recently has it become possible to investigate neural stem cell biology in the AD brain. This is partly because only recent findings revealed the function of adult neural stem cells, but also because animal models for AD have only been available for few years. However, most AD mouse models, which are genetically engineered for Aß deposition, do not develop significant amyloid plaques until past their median lifespan. This limits their availability and the specificity to Aß is reduced due to accompanying age effects. In a first study of this thesis, age related changes of neurogenesis were investigated by monitoring the progressive stages of hippocampal neurogenesis: proliferation, survival and differentiation, in four different age groups of wild type C57BL/6J mice. Net-neurogenesis was rapidly reduced in adult compared to young mice, but remained stable at a low level in aged and senescent mice. This effect could be attributed mostly to an age related decline of proliferation with a concomitant increase of survival rates in aged mice. These results suggest that neurogenesis in aged mice remains as functional as in adult mice, although the plasticity of the neurogenic system appears to be reduced compared to young mice. The finding that a 1 reduced caloric diet, a treatment known to reduce age related defects, did not have an effect on neurogenesis confirmed the finding that neurogenesis is not impaired in aged mice compared to adult mice. In a second study neurogenesis was studied in APP23 mice, a transgenic AD mouse model with progressive amyloid plaque load. Adult Aß pre-depositing and aged Aß high-depositing mice were investigated. Surprisingly, aged APP23 mice showed an increased number of new neurons in the hippocampus compared to age matching controls. For a closer investigation of the interaction of neural stem cells and Aß, we crossed mice expressing GFP under a stem cell specific promoter with a new AD mouse model with cortical plaque deposition in early adulthood. Stem cells were reduced in numbers, strongly attracted to Aß and morphologically altered. In addition, the population of more differentiated immature neurons appeared to be morphologically unaffected by Aß. These findings show that Aß affects neural stem cell biology concomitant with an up-regulation of neurogenesis. Several reports claim that stem cells from the periphery are able to cross the blood brain barrier and are able trans-differentiate to the neuronal lineage. It has also been shown, that the number of cells immigrating from the periphery increases in AD mouse models. Thus, in a third study we investigated if stem cells from the peripheral hematopoietic system could participate in the repair or replacement of the damaged neuronal tissue. APP23 mice were deprived of their immune system by gamma irradiation and later reconstituted with genetically marked hematopoietic stem cells. We found a large number of these cells invading the brains of aged APP23 mice, but cell fate analysis revealed that these cells matured to macrophages or T-cells, but none differentiated towards the neuronal lineage. We conclude that the hematopoietic system is involved in the immune response in the brain, but we found no evidence that it is involved the in repair of the damaged network or in the alterations of neural stem cell biology described above. In conclusion, the results of the present thesis provide evidence of a defective behavior of neural stem cells in the amyloidogenic brain, but also unveil the limitations in the function and ability of neural stem cells in the aged brain. 2 2. Introduction 2.1. Stem Cell Biology Stem cell research has opened one of the most fascinating chapters in the history of biology. Traditionally belonging to the field of developmental biology, stem cells have become of increasing interest for biomedical research in more recent years. Tissue engineering, therapeutic cloning, transgenic animals and gene therapy are among the most discussed applications Background Stem cells are undifferentiated cells that can divide indefinitely. They can either divide symmetrically, producing two identical daughter cells, or asymmetrically producing one identical and one more differentiated daughter cell 1. The least differentiated stem cell type is the omnipotent or totipotent stem cell. It is found in early mammalian embryos (4 8 cell stage) and can form any cell type or tissue including the entire fetus and the placenta 2. The inner cell mass of the blastocyst contains pluripotent stem cells 3. These embryonic stem (ES) cells can be maintained in an undifferentiated state in culture, can differentiate in virtually any kind of cell type, but are not capable of forming an entire embryo. ES cells can be differentiated into multipotent stem cells, which are restricted to their specific lineage. These are hematopoietic, mesenchymal, endodermal or neural stem cells. These lineages follow specific differentiation patterns, with increasingly specialized cells. For example, neuronal stem cells can differentiate into glial-restricted progenitors, motor neuron progenitors, neural crest stem cells and neuron restricted progenitors. By applying the appropriate clues in a defined order it is possible to direct an ES cell towards a specific cell type 4. However, the controversy over using human embryos as a source of these cells have led to intensified research to find ES-like cells in the adult, and to reverse the differentiation process to the pluripotent level 5,6. Although some groups claim to have gained stem cells from skin, bone marrow or hair-follicle, these cells are often restricted to a distinct lineage (for these examples: mesenchymal, hematopoietic or neuronal, respectively). Thus, the only source of ES-like cells from the adult remains somatic cell nuclear transfer (referred to as therapeutic cloning). 3 Adult stem cells For five decades hematopoietic stem cells have been the only adult stem cells known and investigated. Recently it was discovered, that numerous adult tissues contain stem cells. Normally these cells are involved in the homeostatic self-renewal and regenerative processes, but are occasionally activated for repair activity (for review see 7 ). The lumen of the intestine for example is replaced about once a week. Blood and skin is renewed constantly, hair and nails constantly grow. All these systems depend on small local populations of stem cells, which are highly regulated. If the specific program of proliferation, migration and differentiation fails, the respective tissue will either become dysfunctional or cancerous. The arrangement of these proliferative systems is surprisingly conservative for the different tissues where adult stem cells are found. Generally a population of stem cells is harbored in a defined niche. The stem cells proliferate slowly, maintain the size of population and produce another population of transient amplifying precursor cells. These proliferate at a higher rate, and migrate towards the final destination of the specific mature cell type. This results in a differentiation gradient from the stem cell along to the migratory precursor cell to the fully differentiated cell (Fig. 1). Fig. 1: Schematic representation of adult stem cell differentiation. The differentiation process starts by asymmetric division of stem cells in a specific niche and continues during the migration towards the target tissue (green). The intensity of red background signifies the degree of differentiation. 4 2.2. Adult Mammalian Neurogenesis Neurogenesis in the adult brain It has been a common understanding that in postnatal mammals no new neurons are added to the CNS and that any further changes can only be adopted through rewiring of the synaptic connections. In fact this dogma originates from early works at the end of the nineteenth century describing the developmental and adult brain of humans and other mammals These investigators found that the architecture of the brain appears to be fixed soon after birth. At the cellular level, neither mitotic nor developing neurons were observed. Although there were occasional reports on mitotic cells in the brain of adult mammals 11,12 there were no convincing methods to prove that these new cells would differentiate into neurons and be functionally integrated. Using autoradiography to track H 3 Thymidine, incorporated by proliferating cells during mitosis, Joseph Altman published a series of papers in the nineteen sixties showing evidence for adult neurogenesis in the adult rat and cat (for review see 13 ). The scientific community did not recognize the significance of his results. Although Altman s experiments were repeated and combined with electron microscopy, and additional evidence for neurogenesis in songbirds was presented 14,15, the observation of neurogenesis in the adult brain did not get much attention. In the nineteen nineties new techniques emerged. Instead of tritiated Thymidine, Bromodeoxyuridine (BrdU) was used as a proliferation marker. BrdU can easily be labeled with immunohistochemical methods and investigated with brightfield and fluorescence microscopy. In addition specific antibodies against neuronal or glial markers were developed, providing easy methods to distinguish neurons from glia. With the help of these methods adult neurogenesis has been demonstrated to exist until senescence in numerous mammalian species including humans 16. Finally, the neuronal behavior and integration into the network was confirmed by experiments testing long term potentiation (LTP), synapse formation and expression of immediate early genes after stimulation of the hippocampal network Neurogenic regions In mammals three areas of ongoing neurogenesis have been identified: The subventricular zone (SVZ), the olfactory bulb (OB) and the granular cell layer (GCL) in the hippocampus (Fig. 2). Neural stem cells from other brain areas like the neocortex or the thalamus have been isolated in vitro by dissecting these areas and growing the precursors in a growth factor rich medium 21, but in vivo these cells appear to remain quiescent and have not been observed in an activated state. Three subtypes of cells have been identified in the SVZ 22. Astrocyte-like or Type B stem cells cells are positive for glial fibrillary acid protein (GFAP) and most evidence suggests that this is the least differentiated neural stem cell. It has also been shown in embryonic tissue that radial glia cells can differentiate into neural precursor cells and one group was successful in re-differentiating neural precursor cells into radial glia 23. Neural stem cells divide slowly in an asymmetric way, producing astrocyte-like cells and rapidly dividing precursor cells (type C). Type B cells and clusters of type C cells form channels within which the neuroblasts migrate along the sub ventricular zone. The migrating neuroblasts were named type A cells and differentiate from type C daughter cells. The SVZ provides the OB constantly with a stream of progenitor cells, through a path called the rostral migratory stream (Fig. 2). Fig. 2: Sagittal section of a mouse brain showing the neurogenic regions by immunhistochemical staining of immature neurons (DCX). 6 The migratory neuroblasts divide until they integrate as granular cells in the OB. Only very few mature new neurons originating from the SVZ invade the rest of the cortex. This was only found after distinct lesions and not in significant enough numbers to replace the cells lost in the incident 24,25. In the dentate gyrus of the hippocampus there is a similar progression of differentiation, but within a different architecture (Fig. 3). Stem cells located in the subgranular layer produce cluster forming precursors. From there, neuroblasts migrate into the GCL were they extend dendrites into the molecular layer (ML) and send mossy fibers to the CA3 region (Fig. 4). Many more cells are generated than the number that ultimately survives. Following the principle use it or lose it the survival depends on how sufficiently the new cells are activated by incoming neural signals. From the neural stem cell to the mature neuron the cells go through defined steps of division, differentiation, migration and maturation. Using specific markers it is possible to investigate the different phases of development separately (Fig. 3). Fig. 3: Differentiation of neural stem cells into granule cells in the GCL. It is still debated if precursor cells can dedifferentiate to stem cells. The different stages can be monitored by differential expression of specific proteins. Nestin and DCX is co-expressed for a very short time, and DCX and NeuN is not co-expressed. 7 The neurogenic niche Like all adult stem cells, neural stem cells are restricted to a particular niche. Interestingly, in the brain this is closely associated with the angiogenic niche 26. Therefore, neural stem cells are always located close to the vasculature, and even react to the same stimuli as the endothelial stem cells do. This is even more astonishing, as neurogenic and endothelial stem cells are thought to arise from different lineages. However, a recent study reports that neural stem cells have been observed to differentiate into endothelial cells 27. In vitro studies have shown that both endothelial cells and astrocytes can provide various factors to regulate neurogenesis in the vascular niche 28,29, and suppression of neurogenesis by irradiation was accompanied by a destruction of the niche Regulation of neurogenesis Since the general recognition and acceptance of adult neurogenesis a large amount of studies have been conducted to investigate how neurogenesis is regulated. We know that Amphibians and Fish can regenerate neuronal tissue in the retina (for review see 31 ) and findings from the investigations of retinal repair mechanisms have induced a multitude of experiments to study mammalian neurogenesis. The goal is to stimulate the neurogenic capabilities in the brain to induce an endogenous repair mechanism of the damaged mammalian CNS. These studies have revealed a complex regulative interaction of hormones, growth factors, cytokines and neurotransmitters conducted by environmental inputs like physical exercise, enriched environment or learning experiences. Adrenal steroids may be one of the most important neurochemical r
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