Neural tube defects (NTDs) are a world health issue, affecting approximately 1 in every 1000 live births. These congenital defects arise from the improper closure of the neural tube during development, resulting in significant, life-threatening malformations of the central nervous system. Although it has been observed that supplementing women of child-bearing age with folates greatly decreases the chances of having an NTD affected baby, unfortunately these defects still occur. It is accepted that these complex disorders arise from a combination of genetic, environmental, and dietary influences. One such dietary influence is the one-carbon metabolism metabolite, homocysteine. Homocysteine is a byproduct of methylation reactions in the cell that exists in an inverse homeostasis with folate. Homocysteine can also undergo a transformation that allows it to then react with exposed lysine or cysteine residues on proteins, in a process known as N-homocysteinylation or S-homocysteinylation respectively. High levels of homocysteine have been long correlated with many disease states, including NTDs. One potential mechanism by which homocysteine confers its negative effects is through protein N-homocysteinylation. Here, a novel and high-throughput assay for N-homocysteinylation determination is described. This assay is shown to be accurate with mass spectrometry then shown to be biologically relevant using known hyperhomocysteinemia mouse models. This assay was then applied to a cohort of neural tube closure staged mouse embryos with two different genetic mutations that have previously been shown to predispose mice to NTDs. The genotypes explored here are mutations to the LRP6 gene and the Folr1 gene, both of which have been described as folate-responsive NTD mouse models. It was seen that maternal diet and embryonic genotype had the largest influence on the developmental outcome of these embryos; however, the inverse relationship between folate and homocysteine seemed to be established at this early time point, emphasizing the importance of the balance in one-carbon metabolism. One of these genes, LRP6, was then explored in a human cohort of spina bifida cases. Four novel mutations to the LRP6 gene were found and compared to the mouse model used in the previous study. One of the mutations found in the human population was seen to mimic that of the LRP6 mouse model, therefore expanding the potential of this NTD model. / text

Molecular Mechanisms of Hematopoietic Stem Cell Development- The Role of Retinoic Acid Signaling Bhaskar Chanda For the Doctor of Philosophy Medical Biophysics University of Toronto 2013 Abstract During mouse embryonic development, the formation of blood or hematopoiesis occurs in multiple phases. The first phase or primitive hematopoiesis generates a restricted subset of blood cell lineages but is devoid of lymphoid and hematopoietic stem cell (HSC) potential. The next phase of hematopoiesis, also known as definitive hematopoiesis, is characterized by its ability to generate multilineage hematopoietic progenitors and HSCs from a specialized population of endothelial cells known as hemogenic endothelium (HE). Such endothelial to hematopoietic transitions (EHT) have been recently observed at a clonal level, however, molecular mechanisms that underlie EHT leading to the specification of HSCs have remained poorly understood. Here we show that retinoic acid (RA) signaling plays a pivotal role in embryonic hematopoiesis and HSC development. RA signaling inhibits primitive hematopoiesis, and promotes definitive hematopoiesis. This inductive effect of RA signaling extends to the specification of HSCs. Activation of the RA signaling pathway ex vivo in AGM-derived HE dramatically enhanced the repopulating potential, whereas its conditional inhibition in vivo abrogated HSC development. These repressive and inductive effects of RA signaling were mediated primarily via retinoic acid receptor (RAR)- α. We further analyzed the mechanistic basis of RA signaling with a combined use of cellular, molecular and biochemical assays, and show that β-catenin dependent Wnt signaling is the downstream mediator of RA signaling. Collectively, this thesis provides new insight into molecular mechanisms that control embryonic hematopoiesis and identify the RA pathway as a key regulator of definitive hematopoiesis and HSC specification.

Previous research in our laboratory found that inhibiting expression of vimentin, a marker of epithelial-to mesenchymal transition, inhibited cell growth and motility in vitro and in vivo. Tumors derived from vimentin knockdown cells showed features of epithelial redifferentiation and increased expression of differentiation-specific keratins. It is unknown what causes re-expression of keratins when vimentin is inhibited. Although, canonical Wnt signaling may activate NF-κB and repress of keratin and/or induce vimentin expression through β-catenin. We hypothesize that downregulation of differentiation-specific keratins contributes to tumor progression, mediated directly or indirectly by expression of vimentin. Vimentin-negative HN4 cells were transfected with plasmids encoding wild-type, PKCε-phosphomimetic, or unphosphorylatable versions of vimentin. Expression of vimentin was confirmed by western blot and immunofluorescence. Effects on cell growth and motility were determined using MTT, cell proliferation, and wound-closure assays. These results indicate that mutation of vimentin PKCε-phosphorylation sites cause changes in proliferation and filament assembly. Treatment of cells with an NF-κB inhibitor or 5-Aza-C, which allows re-expression of the Wnt inhibitor DKK3, led to a decrease in proliferation. These results suggest that inhibiting Wnt signaling removes the inhibition on GSK-3β and prevents activation of NF-κB, which decreases proliferation.

Retinal degeneration is characterized by the irreversible loss of photoreceptors. A key research question is the identification and characterization of photoreceptor protective factors that prevent or delay vision loss. The Wnt pathway is a critical cellular communication pathway involved in development and diseases of the central nervous system (CNS). Recently, we discovered that multiple components of the Wnt pathway were differentially expressed in the rd1 mouse model of retinal degeneration. One of the most highly upregulated genes was Dickkopf3 (Dkk3), a secreted Wnt pathway protein of unknown function. Additionally, we demonstrated that Wnt signaling is neuroprotective in primary retinal culture (Yi et al., 2007). These data led to the hypothesis that Dkk3 is a regulator of Wnt-mediated neuroprotection during retinal degeneration. The role of Dkk3 in the retina and its activity in the Wnt pathway was identified in this dissertation project using a series of biochemical, molecular and cell biology methodologies. First, Dkk3 was shown to be expressed and secreted from Muller glia in mouse retinal tissue and primary Muller glia culture. I then demonstrated that Muller glia are a Wnt-responsive cell type and that Dkk3 potentiates Wnt3a-mediated signaling. Interestingly, the latter effect was not observed in other cell types in the retina such as retinal ganglion cells and retinal pigmented epithelial cells. Thus, Dkk3 may act on Muller glia to positively modulate Wnt signaling during retinal degeneration, which could potentially amplify the neuroprotective activity of the Wnt pathway. Next, the role of Dkk3 in cellular viability was explored. HEK293 cells stably expressing Dkk3 were shown to be significantly protected from staurosporine-induced apoptosis compared with vector control. This result suggests that Dkk3 may mediate a direct pro-survival effect onto photoreceptors during retinal degeneration. Protein interaction experiments demonstrated that Dkk3 formed a complex with the single pass transmembrane proteins Krm1 and Krm2 in the membrane, potentially in the endoplasmic reticulum (ER). Furthermore, Wnt signaling luciferase reporter assays demonstrated that Krm2, but not Krm1, abolished Dkk3-mediated Wnt3a potentiation. These data suggest that Dkk3 modulates Wnt signaling by antagonizing Dkk1-Krm dependent Wnt inhibition. Further studies will determine whether this activity is sufficient for the potentiation of Wnt signaling by Dkk3. Lastly, co-immunoprecipitation followed by mass spectrometry analysis was used to identify a novel interacting protein of Dkk3. Dkk3 was shown to interact with glucose response protein 78 (GRP78), an ER-resident chaperone. This suggested that Dkk3 protein is part of the unfolded protein response through GRP78 in the ER. In conclusion, these studies identified two novel functions of Dkk3 in regulating Wnt signaling pathway and cellular viability and suggest a physiological role for Dkk3 and Wnt signaling during retinal degeneration. Future studies will explore the significance of Dkk3-Krm and Dkk3-GRP78 interactions in the retina. Further, elucidation of the regulation of Dkk3 and other Wnt ligands in the ER and the consequence of ER stress on the biological activity of Wnt signaling will provide a better understanding of the role of the Wnt pathway during retinal degeneration.

The USP4 is a deubiquitinating enzyme found elevated in certain human lung and adrenal tumours. USP4 has a very close relative, USP15, which has caused great difficulty in studying only one or the other. We have had generated two antibodies specific to USP4 and USP15, and have confirmed that the two do not cross react. Although there have been previous findings of interacting partners, possible substrates and pathways in which it is involved, the biological role of USP4 is mostly unknown. We have used these antibodies to determine that USP4 and USP15 expression differs across tissue and cell types, and that expression changes as the organism ages. We have shown that USP4 plays a role in canonical Wnt signaling, perhaps by stabilizing Beta-catenin, and identified GRK2 as a kinase, phosphorylating USP4. These data have provided enough information to form a hypothesis, implicating USP4 with the destruction complex in the Wnt signaling pathway.

Aggressive fibromatosis is a fibroproliferative tumour that can occur as a sporadic lesion or a manifestation in FAP patients. Tumours are characterized by the stabilization of beta-catenin. Current therapies have yet to offer complete success for primary and recurrent tumours, and there remains a need for more effective therapeutic strategies. In this work, we demonstrate the anti-neoplastic and beta-catenin modulating capacities of Nefopam, a currently approved analgesic agent. We found that Nefopam was able to decrease cell viability and proliferation as well as total beta-catenin levels in human aggressive fibromatosis tumour cells in vitro. Furthermore, Nefopam reduced the number of tumours formed in the Apc+/Apc1638N aggressive fibromatosis mouse model. We also demonstrated that androgens contribute to the development of tumours and could also modulate beta-catenin levels as indicated in Testosterone-treated orchidectomized Apc+/Apc1638N mice. Together, this work suggests that Nefopam and androgen signaling-blocking agents are potential candidates to effectively manage aggressive fibromatosis.

Aggressive fibromatosis is a fibroproliferative tumour that can occur as a sporadic lesion or a manifestation in FAP patients. Tumours are characterized by the stabilization of beta-catenin. Current therapies have yet to offer complete success for primary and recurrent tumours, and there remains a need for more effective therapeutic strategies. In this work, we demonstrate the anti-neoplastic and beta-catenin modulating capacities of Nefopam, a currently approved analgesic agent. We found that Nefopam was able to decrease cell viability and proliferation as well as total beta-catenin levels in human aggressive fibromatosis tumour cells in vitro. Furthermore, Nefopam reduced the number of tumours formed in the Apc+/Apc1638N aggressive fibromatosis mouse model. We also demonstrated that androgens contribute to the development of tumours and could also modulate beta-catenin levels as indicated in Testosterone-treated orchidectomized Apc+/Apc1638N mice. Together, this work suggests that Nefopam and androgen signaling-blocking agents are potential candidates to effectively manage aggressive fibromatosis.

The USP4 is a deubiquitinating enzyme found elevated in certain human lung and adrenal tumours. USP4 has a very close relative, USP15, which has caused great difficulty in studying only one or the other. We have had generated two antibodies specific to USP4 and USP15, and have confirmed that the two do not cross react. Although there have been previous findings of interacting partners, possible substrates and pathways in which it is involved, the biological role of USP4 is mostly unknown. We have used these antibodies to determine that USP4 and USP15 expression differs across tissue and cell types, and that expression changes as the organism ages. We have shown that USP4 plays a role in canonical Wnt signaling, perhaps by stabilizing Beta-catenin, and identified GRK2 as a kinase, phosphorylating USP4. These data have provided enough information to form a hypothesis, implicating USP4 with the destruction complex in the Wnt signaling pathway.

Molecular Mechanisms of Hematopoietic Stem Cell Development- The Role of Retinoic Acid Signaling Bhaskar Chanda For the Doctor of Philosophy Medical Biophysics University of Toronto 2013 Abstract During mouse embryonic development, the formation of blood or hematopoiesis occurs in multiple phases. The first phase or primitive hematopoiesis generates a restricted subset of blood cell lineages but is devoid of lymphoid and hematopoietic stem cell (HSC) potential. The next phase of hematopoiesis, also known as definitive hematopoiesis, is characterized by its ability to generate multilineage hematopoietic progenitors and HSCs from a specialized population of endothelial cells known as hemogenic endothelium (HE). Such endothelial to hematopoietic transitions (EHT) have been recently observed at a clonal level, however, molecular mechanisms that underlie EHT leading to the specification of HSCs have remained poorly understood. Here we show that retinoic acid (RA) signaling plays a pivotal role in embryonic hematopoiesis and HSC development. RA signaling inhibits primitive hematopoiesis, and promotes definitive hematopoiesis. This inductive effect of RA signaling extends to the specification of HSCs. Activation of the RA signaling pathway ex vivo in AGM-derived HE dramatically enhanced the repopulating potential, whereas its conditional inhibition in vivo abrogated HSC development. These repressive and inductive effects of RA signaling were mediated primarily via retinoic acid receptor (RAR)- α. We further analyzed the mechanistic basis of RA signaling with a combined use of cellular, molecular and biochemical assays, and show that β-catenin dependent Wnt signaling is the downstream mediator of RA signaling. Collectively, this thesis provides new insight into molecular mechanisms that control embryonic hematopoiesis and identify the RA pathway as a key regulator of definitive hematopoiesis and HSC specification.

The USP4 is a deubiquitinating enzyme found elevated in certain human lung and adrenal tumours. USP4 has a very close relative, USP15, which has caused great difficulty in studying only one or the other. We have had generated two antibodies specific to USP4 and USP15, and have confirmed that the two do not cross react. Although there have been previous findings of interacting partners, possible substrates and pathways in which it is involved, the biological role of USP4 is mostly unknown. We have used these antibodies to determine that USP4 and USP15 expression differs across tissue and cell types, and that expression changes as the organism ages. We have shown that USP4 plays a role in canonical Wnt signaling, perhaps by stabilizing Beta-catenin, and identified GRK2 as a kinase, phosphorylating USP4. These data have provided enough information to form a hypothesis, implicating USP4 with the destruction complex in the Wnt signaling pathway.