Ischemic stroke related brain damage causes loss of multiple cell types, including neural and vascular cells. The extent of post-stroke neurogenesis and angiogenesis predicts the level of functional regeneration/recovery after stroke. In this regard, my thesis was focused on defining the molecular process that modulates post-stroke functional recovery by co-ordinating post-stroke neurovascular remodeling. Since stroke-related brain damage releases enriched local microenvironmental cues, I examined the role of a signaling-induced epigenetic pathway, an atypical protein kinase C (aPKC)-mediated phosphorylation of CREB Binding Protein (CBP), in regulating post-stroke neurovascular remodeling and functional recovery. This pathway has previously been shown to be activated by metformin, an adenosine monophosphate kinase (AMPK) activator, to promote the differentiation of neural precursors in the developing and adult brain. Here, I first developed a murine focal cortical ischemic stroke model with persistent motor function deficits by combined intra-cortical injections of endothelin-1 (ET-1) and L-NAME into the sensorimotor cortex. Second, I applied the ET-1/L-Name-induced focal cortical stroke model in a knock-in mouse CBPS436A where the aPKC-CBP pathway is deficient, and showed that the aPKC-CBP pathway is involved in post-stroke functional recovery by coordinating neurovascular remodeling. Specifically, CBPS436A-KI mice displayed reduced motor recovery, correlated with reduced vascular remodeling and impaired post-stroke angiogenesis. Intriguingly, I also observed that CBPS436A-KI mice showed a reduction in the population of stroke-induced newborn pericytes but an increase in the population of perivascularly-derived neural precursors, implying that the aPKC-CBP pathway may be involved in the process that reprograms pericytes into neural precursors. Together, this study elucidates the novel role of the aPKC-CBP pathway in modulating neurovascular remodeling and functional recovery following focal ischemic cortical stroke.

The Par complex protein, atypical protein kinase C (aPKC), plays an instrumental role in diverse cell polarities. aPKC is able to restrict substrate localization through a phosphorylation-induced cortical exclusion mechanism, allowing for the generation of molecularly distinct cortical domains. Thus, controlling the localization of aPKC is central to Par-mediated polarity but the mechanism by which aPKC is polarized remains poorly understood. In this dissertation I investigated the restriction of aPKC to the apical cortex of Drosophila neural stem cells, neuroblasts, as these cells dynamically polarize aPKC through repeated asymmetric cell divisions. The polarity created through aPKC phosphorylation must be tightly regulated in order to ensure proper balance between self-renewal and differentiation. To begin, I investigated whether or not aPKC’s so called ‘maturation’ by PDK1 phosphorylation is required for aPKC activity and localization. We found that aPKC’s phosphorylation by PDK1 is required for both polarity and full activity. An aPKC containing an unphosphorylatable activation loop mutation localizes symmetrically around the cortex in a manner independent of its binding partner, Par-6, suggesting that aPKC could interact with the cortex by an unknown mechanism. To investigate how aPKC is able to localize to the cortex independent of Par-6, I used an in vivo structure function analysis of domains within aPKC, accompanied by biochemical approaches. I identified a necessity for the aPKC C1 domain for binding to the neuroblast cortex. This interaction is mediated by negatively charged phospholipids. Neither aPKC interaction, with phospholipids or Par-6, is sufficient to restrict aPKC to the apical cortex. Thus, aPKC polarization utilizes a dual interaction mechanism that takes advantage of both protein-lipid and protein-protein interactions, and proper control of each of these signals is required to prevent neuroblast division defects. One interaction, mediated by the C1, is a general cortical targeting mechanism, whereas the other specifies polarization mediated by Par complex interactions. We conclude that a conformational change induced by these interactions activates aPKC’s catalytic activity, thereby coupling localization and activity. This dissertation includes unpublished co-authored material.

abstract: Cells live in complex environments and must be able to adapt to environmental changes in order to survive. The ability of a cell to survive and thrive in a changing environment depends largely on its ability to receive and respond to extracellular signals. Initiating with receptors, signal transduction cascades begin translating extracellular signals into intracellular messages. Such signaling cascades are responsible for the regulation of cellular metabolism, cell growth, cell movement, transcription, translation, proliferation and differentiation. This dissertation seeks to dissect and examine critical signaling pathways involved in the regulation of proliferation in neural stem cells (Chapter 2) and the regulation of Glioblastoma Multiforme pathogenesis (GBM; Chapter 3). In Chapter 2 of this dissertation, we hypothesize that the mTOR signaling pathway plays a significant role in the determination of neural stem cell proliferation given its control of cell growth, metabolism and survival. We describe the effect of inhibition of mTOR signaling on neural stem cell proliferation using animal models of aging. Our results show that the molecular method of targeted inhibition may result in differential effects on neural stem cell proliferation as the use of rapamycin significantly reduced proliferation while the use of metformin did not. Abnormal signaling cascades resulting in unrestricted proliferation may lead to the development of brain cancer, such as GBM. In Chapter 3 of this dissertation, we hypothesize that the inhibition of the protein kinase, aPKCλ results in halted GBM progression (invasion and proliferation) due to its central location in multiple signaling cascades. Using in-vitro and in-vivo models, we show that aPKCλ functions as a critical node in GBM signaling as both cell-autonomous and non-cell-autonomous signaling converge on aPKCλ resulting in pathogenic downstream effects. This dissertation aims to uncover the molecular mechanisms involved in cell signaling pathways which are responsible for critical cellular effects such as proliferation, invasion and transcriptional regulation. / Dissertation/Thesis / Ph.D. Neuroscience 2014

xi, 93 p. ; ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / The atypical protein kinase C (aPKC) protein has been implicated in several human tumors yet very little is known about how aPKC is regulated. One mechanism that has been proposed as the possible source of several types of tumor is the defective asymmetric cell division of a small number of tumor stem cells. aPKC is required for cell polarization from nematodes to mammals, in tissues as diverse as epithelia, embryonic blastomeres, and neural progenitors. In Drosophila central nervous system, mitotic neural stem cells, termed neuroblasts, recruit the polarity proteins aPKC at the cell apical cortex. pack restricts the localization of the differentiation factors Miranda, Prospero, Brat, and Numb to the cell's basal cortex. Later during mitosis, the cytokinetic furrow sets unevenly about the neuroblast apical-basal axis to produce a large cell (neuroblast) which will continue to divide and self-renew, while the smaller ganglion mother cell inherits differentiation factors and terminally divides to give rise to a pair of neurons and/or glia. Asymmetric cell division is not only critical for generating cellular diversity, it also ensures that a stable population of neural stem cell is constantly maintained while allowing neurogenesis to occur. Despite its conserved role in cell polarity and tumorigenesis, relatively little is known about aPKC regulators and targets. In a co-authored work, I show that the small Rho GTPase, Cdc42, indirectly regulates aPKC. However, this stimulation is modest and the mutant phenotypes are not fully penetrant suggesting that other regulators exist. To isolate other aPKC regulators and targets, I used a biochemical approach to identify aPKC-interacting proteins, and identified one positive regulator and one negative regulator of aPKC. I show that Dynamin-associated protein-160 (Dap160; related to mammalian Intersectin) is a positive regulator of aPKC. I also show that a regulatory subunit of protein phosphatase 2A (PP2A), negatively regulates aPKC. This dissertation includes both my previously published and my co-authored material. / Adviser: Chris Doe

The preimplantation stages of mammalian development are dedicated to the differentiation of two extraembryonic epithelia, the trophectoderm (TE) and the primitive endoderm (PrE), and their segregation from the pluripotent embryonic lineage, the epiblast. The TE and PrE are responsible for implantation into the uterus and for producing the tissues that will support and pattern the epiblast as it develops into the foetus. PrE and epiblast are formed in a two step process that involves random cell fate specification, mediated by fibroblast growth factor (FGF) signalling, and cell sorting through several mechanisms. In the present work I have addressed aspects of both steps of this process. Chimaera assays showed that epiblast precursors transplanted onto a recipient embryo rarely differentiate into PrE, while PrE precursors are able to switch their identity and become epiblast. Transient stimulation or inhibition of the FGF4-ERK pathway in the chimaeras can modify the behaviour of these cells and restore the plasticity of epiblast precursors. This work shows that epiblast precursors are refractory to differentiation signals, thus ensuring the preservation of the embryonic lineage. I have also found that atypical Protein Kinase C (aPKC) is a marker of PrE cells and that pharmacological inhibition of aPKC impairs the segregation of PrE and epiblast precursors. Furthermore, it affects the survival of PrE cells and can alter the subcellular localisation of the PrE transcription factor GATA4. These data indicate aPKC plays a central role for the sorting of the PrE and epiblast populations and links cell position within the embryo to PrE maturation and survival. Lastly, I have found that aPKC can directly phosphorylate GATA4 in vitro. Knockdown of GATA4 affects cell position within the embryo, whereas aPKC knockdown reduces the number of GATA4-positive cells. These results indicate GATA4 plays an important role in cell sorting during preimplantation development and suggest phosphorylation by aPKC could determine its presence in the nuclei of PrE cells. My work, in the light of the current knowledge, supports a model where the earliest cell fate decisions during mammalian development depend on cellular interactions and not on inherited cell fate determinants. This robust mode of development underlies the plasticity of the preimplantation embryo and ensures the formation of the first mammalian cell lineages, critical for any further progression in mammalian development.

Although the mechanisms underlying the divergence of the first cell types in the mouse, the trophectoderm (TE) and the inner cell mass (ICM) have received considerable attention, the upstream signals stimulating their divergence are not well understood. The work presented here examines the roles that morphogenetic factors such as cell adhesion and polarization play in the development of these cell types. I show here that in embryos completely lacking both maternal and zygotic E-cadherin, the normal epithelial morphology of outer cells is disrupted but individual cells still initiate TE and ICM-like fates. A larger proportion of cells than normal expressed TE markers like Cdx2 (a homeodomain containing transcription factor), suggesting that formation of an organized epithelium is not necessary for TE-specific gene expression. Individual cells in such embryos still generate an apical-like domain that correlates with elevated Cdx2 expression. I also show that repolarization can occur in isolated early ICMs from both wild type and Cdx2 mutant embryos, indicating that Cdx2 is not required to initiate polarity. Importantly, I demonstrate a critical role for the Rho-associated kinase ROCK in apical-basal polarization of preimplantation blastomeres. Loss of apical-basal polarization leads to a reduction of Cdx2 expression in outer blastomeres due to activation of Lats1/2 kinase and reduced nuclear Yap1. The influence of polarization upon Lats1/2 kinase is stage-dependent however, as apolar 8-cell blastomeres retain nuclear Yap1. Cell position appears to serve as an additional cue for nuclear localization of Yap and Cdx2 expression from the 8-cell stage to E3.5. Cell polarization plays an additional role in the embryo of maintaining cells in consistently outer or inner positions, thus ensuring that Cdx2 is expressed exclusively in the developing TE. The results of this work demonstrate important links between morphogenesis, cell fate and patterning in the preimplantation embryo. Both cell polarization and cell position act as critical cues to determine gene expression and to pattern this expression within the embryo.

Although the mechanisms underlying the divergence of the first cell types in the mouse, the trophectoderm (TE) and the inner cell mass (ICM) have received considerable attention, the upstream signals stimulating their divergence are not well understood. The work presented here examines the roles that morphogenetic factors such as cell adhesion and polarization play in the development of these cell types. I show here that in embryos completely lacking both maternal and zygotic E-cadherin, the normal epithelial morphology of outer cells is disrupted but individual cells still initiate TE and ICM-like fates. A larger proportion of cells than normal expressed TE markers like Cdx2 (a homeodomain containing transcription factor), suggesting that formation of an organized epithelium is not necessary for TE-specific gene expression. Individual cells in such embryos still generate an apical-like domain that correlates with elevated Cdx2 expression. I also show that repolarization can occur in isolated early ICMs from both wild type and Cdx2 mutant embryos, indicating that Cdx2 is not required to initiate polarity. Importantly, I demonstrate a critical role for the Rho-associated kinase ROCK in apical-basal polarization of preimplantation blastomeres. Loss of apical-basal polarization leads to a reduction of Cdx2 expression in outer blastomeres due to activation of Lats1/2 kinase and reduced nuclear Yap1. The influence of polarization upon Lats1/2 kinase is stage-dependent however, as apolar 8-cell blastomeres retain nuclear Yap1. Cell position appears to serve as an additional cue for nuclear localization of Yap and Cdx2 expression from the 8-cell stage to E3.5. Cell polarization plays an additional role in the embryo of maintaining cells in consistently outer or inner positions, thus ensuring that Cdx2 is expressed exclusively in the developing TE. The results of this work demonstrate important links between morphogenesis, cell fate and patterning in the preimplantation embryo. Both cell polarization and cell position act as critical cues to determine gene expression and to pattern this expression within the embryo.

<p>RhoGTPases are proteins working as molecular switches as they bind and hydrolyze GTP. They are in their active conformation when GTP is bound and are then able to interact with their effector proteins, which relay the downstream signaling. When the GTP is hydrolyzed to GDP, the RhoGTPase is inactivated. RhoGTPases have been shown to be activated by a variety of stimuli and they are implicated in regulation of diverse cellular processes, including cell migration, cell cycle progression, establishment of cell polarity and transformation. </p><p>We identified mammalian Par6 as a novel effector protein for the RhoGTPases Cdc42 and Rac1. The <i>Caenorhabditis elegans</i> homologue of Par6 had previously been shown to be essential for cell polarity development in the worm embryo. We found that endogenous Par6 colocalized with the tight junction protein ZO-1 in MDCKII epithelial cells. Par6 also interacted with mammalian Par3, another member of the <i>par</i> (for partitioning defective) gene family, first identified in <i>C.elegans</i>. Endogenous Par3 also localized to tight junctions in epithelial cells. This suggested that Par6 and Par3 are part of a complex regulating cell polarity also in mammalian cells. The interaction between Par6 and activated Cdc42 and Rac1 suggested a role for these RhoGTPases in the regulation of this complex.</p><p>Co-expression of Par6 together with PKCζ, induced a dramatic change in cell morphology. The cells rounded up and long cellular extensions, resembling neurites, were formed. The ability to induce these changes in cell morphology was found to be dependent on the direct interaction between Par6 and PKCζ, as well as on the kinase activity of PKCζ. We observed that cells co-expressing mPar6C and PKCζ contained bundled microtubules and microtubules that hade been acetylated, indicating that the microtubules were stabilized. </p><p>To investigate the roles of RhoGTPases in PDGF-induced gene expression we performed cDNA microarray analyses on AG01518 human foreskin fibroblasts in which we over-expressed the dominant negative forms of Cdc42, Rac1 and RhoA. We found that the expression of 16 genes, out of the 45 up-regulated by PDGF-BB, were inhibited ≥50% in the presence of dominant negative Cdc42, Rac1 or RhoA. 19 other genes were down-regulated by one or two of the dominant RhoGTPases. Our data implied that the expression of many PDGF-BB induced genes can be affected by RhoGTPase signaling. </p><p>In conclusion, the work presented here has increased the knowledge of the involvement of RhoGTPase signaling in establishment of cell polarity and gene regulation.</p>

RhoGTPases are proteins working as molecular switches as they bind and hydrolyze GTP. They are in their active conformation when GTP is bound and are then able to interact with their effector proteins, which relay the downstream signaling. When the GTP is hydrolyzed to GDP, the RhoGTPase is inactivated. RhoGTPases have been shown to be activated by a variety of stimuli and they are implicated in regulation of diverse cellular processes, including cell migration, cell cycle progression, establishment of cell polarity and transformation. We identified mammalian Par6 as a novel effector protein for the RhoGTPases Cdc42 and Rac1. The Caenorhabditis elegans homologue of Par6 had previously been shown to be essential for cell polarity development in the worm embryo. We found that endogenous Par6 colocalized with the tight junction protein ZO-1 in MDCKII epithelial cells. Par6 also interacted with mammalian Par3, another member of the par (for partitioning defective) gene family, first identified in C.elegans. Endogenous Par3 also localized to tight junctions in epithelial cells. This suggested that Par6 and Par3 are part of a complex regulating cell polarity also in mammalian cells. The interaction between Par6 and activated Cdc42 and Rac1 suggested a role for these RhoGTPases in the regulation of this complex. Co-expression of Par6 together with PKCζ, induced a dramatic change in cell morphology. The cells rounded up and long cellular extensions, resembling neurites, were formed. The ability to induce these changes in cell morphology was found to be dependent on the direct interaction between Par6 and PKCζ, as well as on the kinase activity of PKCζ. We observed that cells co-expressing mPar6C and PKCζ contained bundled microtubules and microtubules that hade been acetylated, indicating that the microtubules were stabilized. To investigate the roles of RhoGTPases in PDGF-induced gene expression we performed cDNA microarray analyses on AG01518 human foreskin fibroblasts in which we over-expressed the dominant negative forms of Cdc42, Rac1 and RhoA. We found that the expression of 16 genes, out of the 45 up-regulated by PDGF-BB, were inhibited ≥50% in the presence of dominant negative Cdc42, Rac1 or RhoA. 19 other genes were down-regulated by one or two of the dominant RhoGTPases. Our data implied that the expression of many PDGF-BB induced genes can be affected by RhoGTPase signaling. In conclusion, the work presented here has increased the knowledge of the involvement of RhoGTPase signaling in establishment of cell polarity and gene regulation.