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The dentate gyrus (DG) plays a crucial role in hippocampal-related memory. The most abundant cellular type in the DG, namely granule neurons, are developmentally generated around postnatal day P6 in mice. Moreover, a unique feature of the DG is the occurrence of adult hippocampal neurogenesis, a process that gives rise to newborn granule neurons throughout life. Adult-born and developmentally generated granule neurons share some maturational aspects but differ in others, such as in their positioning within the granule cell layer. Adult hippocampal neurogenesis encompasses a series of plastic changes that modify the function of the hippocampal trisynaptic network. In this regard, it is known that glycogen synthase kinase 3ß (GSK-3ß) regulates both synaptic plasticity and memory. By using a transgenic mouse overexpressing GSK-3ß in hippocampal neurons, we previously demonstrated that the overexpression of this kinase has deleterious effects on the maturation of newborn granule neurons. In the present study, we addressed the effects of GSK-3ß overexpression on the morphology and number of dendritic spines of developmentally generated granule neurons. To this end, we performed intracellular injections of Lucifer Yellow in developmentally generated granule neurons of wild-type and GSK-3ß-overexpressing mice and analyzed the number and morphologies of dendritic spines (namely, stubby, thin and mushroom). GSK-3ß overexpression led to a general reduction in the number of dendritic spines. In addition, it caused a slight reduction in the percentage, head diameter and length of thin spines, whereas the head diameter of mushroom spines was increased.

Glycogen synthase kinase-3b (GSK-3b) is a serine-threonine kinase implicated in multiple processes and signaling pathways. Its dysregulation is associated with different pathological conditions including Alzheimer’s disease (AD). Here we demonstrate how changes in GSK-3b activity and/or levels regulate the production and subsequent secretion of fractalkine, a chemokine involved in the immune response that has been linked to AD and to other different neurological disorders. Treatment of primary cultured neurons with GSK-3b inhibitors such as lithium and AR-A014418 decreased full-length fractalkine in total cell extracts. Opposite effects were observed after neuron transduction with a lentiviral vector overexpressing the kinase. Biotinylation assays showed that those changes mainly affect the plasma membrane-associated form of the protein, an observation that positively correlates with changes in the levels of its soluble form. These effects were confirmed in lithium-treated wild type (wt) mice and in GSK-3b transgenic animals, as well as in brain samples from AD patients, evident as age-dependent (animals) or Braak stage dependent changes (humans) in both the membrane-bound and the soluble forms of the protein. Further immunohistochemical analyses demonstrated how GSK-3b exerts these effects by affecting the trafficking of the chemokine from the Golgi to the plasma membrane, in different and opposite ways when the levels/activity of the kinase are increased or decreased. This work provides for the first time a mechanism linking GSK-3b and fractalkine both in vitro and in vivo, with important implications for neurological disorders and especially for AD, in which levels of this chemokine might be useful as a diagnostic tool.

Umbilical cord blood (UCB) is a well established alternative source of haematopoietic stem/progenitor cells (HSPC) for haematopoietic stem cell transplantation (HSCT). One factor limiting the wider use of UCB for transplantation is the relatively low cell dose from a UCB unit compared to bone marrow (BM) and peripheral blood (PB). To overcome this barrier, many strategies have been tested to expand HSPC numbers ex vivo. The aims of this thesis were to define the phenotypic, functional and transcriptional changes that occur during ex vivo expansion of UCB HSPC and investigate how activation of the Wingless (Wnt) pathway via GSK-3 inhibition can provide a novel approach to improve transplantation outcomes using expanded cells. In vitro and in vivo models of HSPC function were used to evaluate the impact of expansion and GSK-3 inhibition on progenitor function while gene array technology was used to investigate potential molecular mechanisms.We have shown that expansion of UCB HSPC results in a loss of primitive phenotype, an increase in progenitors with reduced function and a shift in transcriptional profile from ‘stem-like quiescent’ to ‘divide and differentiate’. Brief treatment of expansion cultures with a GSK-3 inhibitor resulted in delayed cell cycle progression which translated into improved PB and BM repopulation. Gene expression analysis revealed modulation of Notch and Tie2 pathways, suggesting their potential role in improving stem cell engraftment. In addition, down-regulation of cyclin D1 and upregulation of CDK inhibitor p57 known to be important in maintenance of quiescence was observed.This work is a timely reminder of the medical revolution that E. Donnall Thomas pioneered in the 1950’s, paving the way for successful treatment of those with haematological malignancies and other blood disorders. In addition, the year 2013 marked the 25th anniversary of the first UCB transplant that took place in France in October 1988. Since then over 600,000 UCB uni