Description of the cell cycle features of neural progenitors during late stages of neurogenesis in a gyrencephalic mammal, the ferret.

G-CSF is routinely used to treat neutropenia/leukopenia or to increase hematopoietic stem cell generation in bone marrow donors. G-CSF and its receptor, G-CSFR, are produced by various cell types both in the peripheral circulation and within brain. As a consequence, exogenous administration of G-CSF results in a broad spectrum of effects involving hematopoietic, immune and central nervous systems. G-CSF administration in a mouse model of Alzheimer's disease (AD) has revealed both cognitive benefits and disease modifying effects: a) decreased Aβ plaque burden, b) increased microgliosis, c) increased neurogenesis and d) improved performance in radial arm water maze (RAWM). In clinical studies, G-CSF plasma levels were found to be lower in patients with early AD in comparison to healthy age matched controls. A course of G-CSF administration in humans is known to increase levels of circulating hematopoietic stem cells (CD34 cells), monocytes and neutrophils in patients with neutropenia and when administered to patients with AD, there is also a similar increase in absolute monocyte count, CD34 cells and total neutrophils. The extent to which the beneficial effects of G-CSF in AD depend on monocyte infiltration into CNS, compared to direct neurotrophic actions of G-CSF on the CNS, is not known. The overall goal of this study was to investigate and understand the effects of G-CSF in an AD mouse model, but more specifically to distinguish the actions of G-CSF that affect the peripheral monocyte population from the direct actions on CNS. The first approach was to examine in vitro effects of G-CSF within a monocytic cell line (THP-1) and a neuronal cell line (SH-SY5Y). The second approach was to study effects of G-CSF on infiltration of bone marrow-derived cells into the brain by utilizing a chimeric GFP+ APP/PS1 AD mouse model. The third approach was to assess the effects of G-CSF on hippocampal neurogenesis in both a wild-type and AD mouse model. Comparison of the monocytic and neuronal cell lines showed a) G-CSF interacts with its cognate receptor with different binding kinetics and with a greater affinity for the monocyte G-CSFR, b) the number of G-CSF receptors in neurons is greater than in monocytes, and c) the anti-apoptotic response in neurons occurs at lower concentrations of G-CSF than in monocytes. Various concentrations of G-CSF increased proliferation of both the monocytic and neuronal cell line in vitro. G-CSF did not improve migratory properties of the monocytic cell line, either adhesiveness or migration through a membrane. In vivo G-CSF treatment (250μg/kg s.c. qod for 2 ½ weeks) in both the AD chimeric and non-chimeric AD mice resulted in increased microgliosis and decreased amyloid plaque burden in the hippocampus. In the chimeric AD mice, G-CSF treatment did not increase infiltration of GFP+ bone marrow derived cells (BMDC) into brain parenchyma and did not increase adhesion to microvasculature. In the non-chimeric AD mice there was improvement of neurogenesis to non-transgenic levels after G-CSF treatment and an increase in synaptogenesis in the CA1 region of the hippocampus. The effects of G-CSF on the endogeneous microglial population are most likely responsible for the increase in microgliosis, as no significant increase of BMDC infiltration into the brain parenchyma was found in vivo. The enhanced proliferation and improved viability of the neuronal cell line after G-CSF treatment may explain the improvement in neurogenesis and significant increase in synaptogenesis seen in the AD mouse model. The actions of G-CSF on neural stem/progenitor cells to stimulate hippocampal neurogenesis and to enhance resident microglial capacity to decrease amyloid burden are the most likely mechanisms responsible for the behavioral improvement seen in the AD mouse model.

Olfactory sensory neurons (OSNs) undergo continual degeneration and replacement throughout life, a cycle that can be synchronized experimentally by performing olfactory bulbectomy (OBX). OBX induces apoptosis of mature OSNs, which is followed by an increase in the proliferation of progenitor basal cells. Macrophages, functionally diverse immune effector cells, phagocytose the apoptotic OSNs and regulate the proliferation of basal cells. This provides an advantageous environment to study how macrophages regulate neuronal death, proliferation, and replacement. The purpose of this dissertation was to identify the cellular and molecular mechanisms by which macrophages regulate the degeneration/proliferation cycle of OSNs. Macrophages were selectively depleted using liposome-encapsulated clodronate (Lip-C). Intranasal and intravenous administration of Lip-C decreased the number of macrophages in the OE of sham and OBX mice by 38% and 35%, respectively, compared to mice treated with empty liposomes (Lip-O). Macrophage depletion significantly decreased OE thickness (22% and 21%, p<0.05), the number of mature OSNs (1.2- and 1.9-fold, p<0.05), and basal cell proliferation (7.6- and 3.8-fold, p<0.05) in sham and OBX mice, respectively, compared to Lip-O mice. Additionally, at 48 h following OBX, OSN apoptosis increased significantly (p<0.05) in the OE of Lip-C mice compared to Lip-O mice. A microarray analysis was performed to identify the genomic changes underlying the cellular changes associated with macrophage depletion. There were 4,024 genes with either a significant interaction between group (Lip-C vs. Lip-O) and treatment (OBX vs. sham) or a significant main effect. There were a number of significantly regulated immune response and cytoskeletal genes, and genes encoding neurogenesis regulators and growth factors, most of which were expressed at lower levels in Lip-C mice compared to Lip-O mice. Sdf1, the ligand for the chemokine receptor Cxcr4 involved in leukocyte trafficking, axon guidance, and cell migration, was localized to macrophages on the protein level. Additionally, the microarray expression pattern of Hdgf, a growth factor that promotes neuronal survival and proliferation, was validated on the protein level using immunohistochemistry. HDGF appeared to be localized to basal cells and OSNs where it could act as a proliferative or survival factor whose expression is regulated in part by macrophages.

The Hedgehog (Hh) signalling pathway is one of the key signalling pathways orchestrating intricate organogenesis, including the development of neural tube, heart and skeletal muscle. Yet, insufficient mechanistic understanding of its diverse roles is available. Here, we show the molecular mechanisms regulating the neurogenic, cardiogenic and myogenic properties of Hh signalling, via effector protein Gli2, in embryonic and adult stem cells. In Chapter 2, we show that Gli2 induces neurogenesis, whereas a dominant-negative form of Gli2 delays neurogenesis in P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (ES) cell model. Furthermore, we demonstrate that Gli2 associates with Ascl1/Mash1 gene elements in differentiating P19 cells and activates the Ascl1/Mash1 promoter in vitro. Thus, Gli2 mediates neurogenesis in P19 cells at least in part by directly regulating Ascl1/Mash1 expression. In Chapter 3, we demonstrate that Gli2 and MEF2C bind each other’s regulatory elements and regulate each other’s expression while enhancing cardiomyogenesis in P19 cells. Furthermore, dominant-negative Gli2 and MEF2C proteins downregulate each other’s expression while imparing cardiomyogenesis. Lastly, we show that Gli2 and MEF2C form a protein complex, which synergistically activates cardiac muscle related promoters. In Chapter 4, we illustrate that Gli2 associates with MyoD gene elements while enhancing skeletal myogenesis in P19 cells and activates the MyoD promoter in vitro. Furthermore, inhibition of Hh signalling in muscle satellite cells and in proliferating myoblasts leads to reduction in MyoD and MEF2C expression. Finally, we demonstrate that endogenous Hh signalling is important for MyoD transcriptional activity and that Gli2, MEF2C and MyoD form a protein complex capable of inducing skeletal muscle-specific gene expression. Thus, Gli2, MEF2C and MyoD participate in a regulatory loop and form a protein complex capable of inducing skeletal muscle-specific gene expression. Our results provide a link between the regulation of tissue-restricted factors like Mash1, MEF2C and MyoD, and a general signal-regulated Gli2 transcription factor. We therefore provide novel mechanistic insights into the neurogenic, cardiogenic and myogenic properties of Gli2 in vitro, and offer novel plausible explanations for its in vivo functions. These results may also be important for the development of stem cell therapy strategies.

The classical cell cycle regulatory pathway is well appreciated as a key regulator of cell fate determination during neurogenesis; however, the extent of pRB/E2F function in neural stem and progenitor cells is not fully understood, and insight into the mechanisms underlying its connection with cell fate regulation are lacking. The E2F3 transcription factor has emerged as an important regulator of neural precursor cell (NPC) proliferation in the embryonic and adult forebrain, and we demonstrate here that it also influences the self-renewal potential of NPCs. Using knockout mouse models of individual E2F3 isoforms, we demonstrate the surprising result that the classical transcriptional activator E2F3a represses NPC self-renewal and promotes neuronal differentiation, while E2F3b promotes the expansion of the NPC pool and inhibits differentiation. We attribute these opposing activities to a unique mechanism of transcriptional regulation at the Sox2 locus, a key regulator of stem cell pluripotency, whereby E2F3a recruits transcriptional repressors to this site, and E2F3b promotes Sox2 activation. Importantly, E2F3a-mediated Sox2 regulation is necessary for cognitive function in the adult. Additionally, through the determination of genome-wide promoter binding sites for E2f3 isoforms as well as E2F4, another key regulator of NPC self-renewal, we determined that E2Fs are poised to regulate an extensive set of target genes with key roles in regulating diverse cell fate choices in NPCs, including self-renewal, cell death, progenitor expansion, maintenance of the precursor state, and differentiation. Together, these results reveal a diversity of function for E2Fs in the control of neural precursor cell fate, and identify E2F3 isoforms as important regulators of the pluripotency and stem cell maintenance gene Sox2.

The dentate gyrus is a region that hosts most of the hippocampal cells in mammals. Nevertheless, its role in spatial memory remains poorly understood, especially in light of the recently-studied phenomenon of adult hippocampal neurogenesis and its possible role in aging and chronic brain disease. We found that chronic over-expression of transforming growth factor beta1 (TGFbeta1), a cytokine involved in neurodegenerative disease, results in several modifications of brain structure, including volumetric changes and persistent astrogliosis. Furthermore, TGFbeta1 over-expression affects the generation of new neurons, leading to an increased number of neurons in the dentate gyrus and deficits in spatial memory acquisition and storage in aged mice. Nonetheless, reducing neurogenesis via pharmacological treatment impairs spatial memory in juvenile mice but not in adult or aged mice. This suggests that the addition of new cells to hippocampal circuitry, and not the increased plasticity of these cells, is the most relevant role of neurogenesis in spatial memory. We tested this idea by modifying proliferation in the dentate gyrus at several ages using multiple techniques and evaluating the incorporation of newborn neurons into hippocampal circuitry. We found that all granule neurons, recently generated or not, have the same probability of being incorporated. Therefore, the number of new neurons participating in memory circuits is proportional to their availability. Our conclusion is that adult-generated cells have the same functional relevance as those generated during development. Together, our data show that the dentate gyrus is important for memory processing and that adult neurogenesis may be relevant to its functionality by optimizing the number of neurons for memory processing. The equilibrium between neurogenesis and optimal dentate gyrus size is disrupted when TGFbeta1 is chronically increased, which occurs in neurodegenerative pathologies, leading to cognitive impairment in aged animals.

We have used the chicken retina as a model for investigating cell cycle regulation and cell fate commitment during central nervous system development. This thesis focuses on the characterization of and commitment to the horizontal cell fate in the retina. Horizontal cells are interneurons that provide intraretinal signal processing prior to information relay to the brain. We have identified molecular markers that selectively distinguish the three subtypes of horizontal cells, previously described in the chicken retina based on morphology. Subtype specific birth-dating revealed that horizontal cell subtypes are generated consecutively by biased progenitors that are sensitive to the inhibitory effects of follistatin. Follistatin stimulates proliferation in progenitors by repressing the differentiation signal of activin. Initially, injection of follistatin led to a decrease in committed horizontal cells but as the inhibitory effect dissipated it resulted in an increased number of horizontal cells. During development committed horizontal cell progenitors migrate to the vitreal side of the retina where they become arrested in G2-phase for approximately two days. When the arrest is overcome the horizontal cell progenitors undergo ectopic mitosis followed by migration to their designated layer. The G2-phase arrest is not triggered or maintained by any of the classic G2-arrest pathways such as DNA damage or stress. Nevertheless, we show that the cyclin B1-Cdk1 complex has a central role in maintaining this G2-phase arrest. Two transcription factors, FoxN4 and Ptf1a, are required for the generation of horizontal cells. We show that these factors are also sufficient to promote horizontal cell fate. Overexpression of FoxN4 and Ptf1a resulted in an overproduction of horizontal- and amacrine cells at the expense of ganglion- and photoreceptor cells. We identified Atoh7, a transcription factor required for the generation of ganglion cells, as a Ptf1a transcriptional target for downregulation. Our data support a common horizontal/amacrine lineage separated from the ganglion/photoreceptor lineage by the action of Ptf1a. In conclusion, these data describe several novel characteristics of horizontal cells enhancing our understanding of neural development and cell fate commitment.

Neurogenesis occurs in exclusive regions in the adult nervous system, the subventricular zone and dentate gyrus in the brain, and olfactory epithelium (OE) in the periphery. Cell replacement after death or injury, occurs to varying degrees in neural tissue, and is thought to be dependent upon the biological responses of stem and/or progenitor cells. Despite the progress made to identify adult OE and central nervous system (CNS) progenitors and lineage trace their progeny, our spatial and temporal understanding of embryonic OE neuroglial progenitors has been stalled by the paucity of identifiable genes able to distinguish individual candidate progenitors. In the developing CNS, radial glia serve as both neural progenitors and scaffolding for migrating neuroblasts and are identified by the expression of a select group of antigens, including nestin. Here, I show that the embryonic OE contains a novel radial glial-like progenitor (RGLP) that is not detected in adult OE. RGLPs express the radial glial antigens nestin, GLAST and RC2, but not brain lipid binding protein (BLBP), which, distinct from CNS radial glia, is instead found in olfactory ensheathing cells, a result confirmed using lineage tracing with BLBP-cre mice. Nestin-cre-mediated lineage tracing with three different reporters reveals that only a subpopulation of nestin-expressing RGLPs activate the “CNS-specific” nestin regulatory elements, and produce spatially restricted neurons in the OE and vomeronasal organ. The dorsal-medial restriction of transgene-activating cells is also seen in the embryonic OE of Nestin-GFP transgenic mice, where GFP is found in a subpopulation of GFP+ Mash1+ neuronal progenitors, despite the fact that endogenous nestin expression is found in RGLPs throughout the OE. In vitro, embryonic OE progenitors produce three biologically distinct colony subtypes, that when generated from Nestin-cre/ZEG mice, produce GFP+ neurons, recapitulating their in vivo phenotype, and are enriched for the most neurogenic colony subtype. Neurogenesis in vitro is driven by the proliferation of nestin+ progenitors in response to FGF2. I thus provide evidence for a novel neurogenic precursor, the RGLP of the OE, that can be regulated by FGF2, and provide the first evidence for intrinsic differences in the origin and spatiotemporal potential of distinct progenitors during OE development.

Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.