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Medulloblastomas represent the most frequent malignant brain tumour in children, and are thought to develop in the posterior fossa of the cerebellum. Some patients with medulloblastomas have a deficiency in the tumour suppressor gene Apc (Turcot’s syndrome). Although the majority of medulloblastomas arise sporadically, people with Apc mutations are 92 times more likely to develop medulloblastomas. Apc encodes a very large protein known to function as a regulator of the Wnt signalling pathway. Activation of the canonical Wnt pathway leads to the stabilisation of beta-catenin. In response to Wnt signals, beta-catenin translocates to the nucleus where it interacts with the LEF/TCF family of transcription factors to activate transcription of target genes such as c-myc and cyclinD1. Mutations of Apc that cause an increase in beta-catenin are found to be tumourgenic, whereas other mutations are not. Therefore it is thought that the main tumour suppression function of Apc is in its ability to destabilise and hence reduce cytoplasmic beta-catenin. The central hypothesis of this thesis is that the loss of Apc can lead to the development of medulloblastoma. Work from other groups has reported activation of Wnt signalling in a proportion of primary medulloblastomas. We undertook a study to assess this by using the cre-loxP recombination system to mutate Apc in a temporal and spatial manner. This approach is necessary as Apc has many functions in development and Apc mutant mice (Apcmin) do not develop past embryonic day 6.5 (E6.5). To date, there are no known cre-strains available to mutate Apc specifically in the cerebellum at early postnatal stages, so we combined the creloxP method with an avian retrovirus mediated method for tissue specific gene delivery (RCAS/tv-a system), in an attempt to create a strain of mice which carried the genotype Ntv-a +;ApcLoxP/LoxP. This would allow us to infect an RCAS-cre virus directly into the hindbrain at postnatal day 4 (P4). However subsequent genotyping of these animals showed that none carried the desired genotype of Ntv-a +;ApcLoxP/LoxP, making it impossible for both copies of Apc to be mutated in a mouse most likely because both the Ntv-a and Apc transgenes were located on the same chromosome. Consistent with this, out of a total of 265 mice none were found to have the Ntv-a +;ApcLoxP/LoxP genotype. We then adopted an alternative method for mutating Apc by infecting ApcLoxP/LoxP mice directly with an AdCre virus. PCR analysis showed that Apc was mutated, however the AdCre virus did not infect cells of the cerebellum, and instead only infected the choroid plexus. In these animals, 7 of 94 (7%) developed hydrocephalus indicating that losing Apc in the choroid plexus may promote hydrocephalus. Finally, to address the role of Apc in normal hindbrain development, we crossed our ApcLoxP/LoxP mice to an En1cre strain which caused mutation in Apc from E8.5 in the midhindbrain region. The resulting En1cre+;ApcLoxP/LoxP mutants displayed hydrocephalus in all ventricles and an in-growth of mesenchyme tissue at the mid-hindbrain border, closely associated with a tumour-like area of cells showing activated Wnt signalling. No mice were found to live past E18.5. In conclusion, the role of Apc in medulloblastoma remains unclear. Future studies could use a different technique to mutate Apc such as crossing ApcLoxP/LoxP mice to the new nestin-creER strain and inducting cre with administration of tamoxifen.

Medulloblastomas are tumours of cerebellar origin and are thought to arise from the malignant transformation of progenitor cells in the developing cerebellum. A number of developmental signalling pathways are required for the precise cell specification, proliferation, migration and differentiation involved in forming the mature cerebellum and it is the dysregulation of these processes that can lead to the eventual formation of a tumour. Genes encoding components of the canonical Wnt/β-catenin signaling pathway are mutated in around 15% of medulloblastomas and germline mutations that activate this pathway are known to predispose to medulloblastoma. Despite this, the contribution of Wnt/β-catenin signaling to normal cerebellum development is not yet well understood and the developmental origins of medulloblastoma arising from activation of this pathway are only beginning to be revealed. Therefore, the aims of this thesis were to characterise the spatio-temporal nature of Wnt/β-catenin signalling during cerebellum development and to investigate its function, with the broad goal of informing our understanding of how medulloblastoma arises from oncogenic activation of Wnt/β-catenin signalling. To address the first aim I utilised a LacZ expressing Wnt/β-catenin signalling reporter mouse to characterize the spatio-temporal pattern of Wnt/β-catenin pathway activation during cerebellum development. Analysis of LacZ reporter expression revealed a pattern of transient Wnt/β-catenin activity in discrete cell populations throughout cerebellum development. I found that Wnt/β-catenin activity is present during the early specification of granule cells at the cerebellar rhombic lip but not during the expansion of this cell population at later stages. During perinatal development Wnt/β-catenin activity shifts to the cerebellar ventricular zone, a known germinal centre for GABAergic interneurons and glia, and was observed in cells radiating out from this region. By early postnatal development the expression of the Wnt/β-catenin reporter became progressively restricted to the developing Bergmann glia population. To investigate the function of Wnt/β-catenin in these cell lineages and how its dysregulation could contribute to medulloblastoma, I used a combination of ex vivo organotypic culture, in utero electroporation and tissue-specific gene targeting to manipulate components of the pathway. Culturing slices of E18.5 cerebellum in the presence of small molecule activators of the Wnt/β-catenin pathway revealed a reduction in the expression of glial markers Sox9 and GFAP. In addition, interneuron lineage marker Pax2 was also reduced, supporting the conclusion that dysregulation of Wnt/β-catenin signalling affects the generation of cell lineages from the ventricular zone. To investigate this hypothesis further, I constitutively activated the Wnt/β-catenin signalling pathway in the developing cerebellum using Cre-Lox gene targeting to knock out Apc, a negative regulator of the pathway, in ventricular zone derived lineages. Cre-induced recombination of Apc resulted in nuclear accumulation of β-catenin, a sign that the pathway had become ectopically activated. Furthermore, a reduction in the expression of Sox9 and Pax2 was also observed in these mutant cells. From these data, I conclude a potential role for Wnt/β-catenin signaling in the regulation of glial/interneuron progenitors. Combined, these data support a model where Wnt/β-catenin signalling could perform multiple functions in specification of the granule lineage, regulation of glial/interneuron progenitors and in glial differentiation/maturation. Importantly, dysregulation of progenitor self-renewal and differentiation is widely acknowledged to promote tumourigenesis. Thus, the data in this thesis support a potential mechanism for the development of medulloblastoma from the dysregulation of ventricular zone progenitors.

Herpes simplex viruses (HSV) establish latent infections in sensory neurons of their host and are maintained in this state by little understood mechanisms that, at least in part, are regulated by signalling through nerve growth factor (NGF) and its receptor tropomyosin related kinase, trkA. Previous studies have demonstrated that Zhangfei is a transcriptional factor that is expressed in differentiated neurons and is thought to influence HSV replication and latency. Zhangfei, like the HSV trans-activator VP16 and Luman, binds the ubiquitous nuclear protein host cell factor (HCF) inhibiting the ability of VP16 and Luman to initiate HSV replication. <p>Recently, Brn3a, another neuronal factor thought to influence HSV latency and reactivation was found to possess an HCF-binding domain and could potentially require HCF for activity. The neuronal POU IV domain protein, Brn3a, among its many regulatory functions has been described as an enhancer of the NGF receptor trkA, during development in mouse. I therefore investigated the possible link between Brn3a, TrkA, NGF signaling, HCF, Zhangfei and HSV-1 latency and reactivation. I hypothesized that Zhangfei would also suppress the ability of Brn3a to activate the expression of TrkA and that this would have an impact on NGF-TrkA signaling and, consequently on HSV-1 reactivation from latency.<p>My first study determined which Brn3a/trkA promoter interactions were important for trkA transcription. I constructed a plasmid that contains 1043 base pairs of genomic sequences that extend from 30 nucleotides upstream of trkA coding region. In contrast to previous data, a short 190 bp region that lies proximal to the trkA initiation codon was sufficient for Brn3a trans-activation in NGF-differentiated PC12, Vero and human medulloblastoma cells. At least two portions of the 190 bp fragment bind to Brn3a. In addition, Brn3a increased endogenous levels of trkA transcripts in PC12 cells and initiated trkA expression in medulloblastoma cells, which normally do not express trkA. <p>The second step was to determine the effects of HCF and Zhangfei association with Brn3a on trkA trans-activation. I found that Brn3a required HCF for activating the trkA promoter and that Zhangfei has a suppressive effect over Brn3a-trkA activation in non-neuronal cells. In sympathetic neuron-like NGF-treated PC12 cells, Zhangfei did not suppress the ability of Brn3a to activate the TrkA promoter, however, Zhangfei was able capable of inducing the expression of TrkA in the absence of Brn3a. Both Brn3a and Zhangfei induced the expression of endogenous trkA in PC12 cells.<p>Since Vero and PC12 cells are not from human origin I wanted to examine the ability of Zhangfei to induce trkA transcription in medulloblastoma cells, that because of its tumor nature do not express trkA. TrkA transfections in these cells have shown to drive them to cell arrest or apoptosis. Since Zhangfei is not express in medulloblastoma tumors I then used ONS-76 medulloblastoma cells as a model to determine Zhangfeis envolvement in the NGF-trkA signaling pathway.<p> I show herein that in ONS-76 medulloblastoma cells resveratrol, an inducer of apoptosis and differentiation, increased the expression of Zhangfei and trkA as well as Early Growth Response Gene 1 (Egr1), a gene normally activated by NGF-trkA signalling. ONS-76 cells stop growing soon after treatment with resveratrol and a portion of the cell undergo apoptosis. While the induction of Zhangfei in resveratrol-treated cells was modest albeit consistent, the infection of actively growing medulloblastoma cells with an adenovirus vector expressing Zhangfei mimicked the effects of resveratrol. Zhangfei activated the expression of trkA and Egr1 and caused these cells to display markers of apoptosis. The phosphorylation of Erk1, an intermediate kinase in the NGF-trkA signaling critical for differentiation, was observed in Zhangfei infected cells, supporting the hypothesis that Zhangfei is a mediator of trkA-NGF signaling in theses cells leading either to differentiation or apoptosis. Binding of HCF by Zhangfei did not appear to be required for this effect as a mutant of Zhangfei incapable of binding HCF was also able to induce the expression of trkA and Egr1. <p>In in vivo and in vitro models of HSV-1 latency, the virus reactivates when NGF supply to the neuron is interrupted. Based on the above evidence Zhangfei, in HSV-1 latently infected neurons, would have the ability to prolong a state of latency by inducing trkA expression allowing the activation of NGF-trkA signaling pathway. Since NGF is produced by many cell types it is possible that reactivation is triggered not by a decrease in NGF but by a down-regulation of TrkA expression.Therefore, if Zhangfei expression is suppress the trkA signaling could be interrupted or shifted towards apoptosis signaling, this would allow neuronal HCF-binding proteins like Luman, which can activate HSV IE expression, to initiate HSV IE expression and subsequently viral replication.

Herpes simplex viruses (HSV) establish latent infections in sensory neurons of their host and are maintained in this state by little understood mechanisms that, at least in part, are regulated by signalling through nerve growth factor (NGF) and its receptor tropomyosin related kinase, trkA. Previous studies have demonstrated that Zhangfei is a transcriptional factor that is expressed in differentiated neurons and is thought to influence HSV replication and latency. Zhangfei, like the HSV trans-activator VP16 and Luman, binds the ubiquitous nuclear protein host cell factor (HCF) inhibiting the ability of VP16 and Luman to initiate HSV replication. <p>Recently, Brn3a, another neuronal factor thought to influence HSV latency and reactivation was found to possess an HCF-binding domain and could potentially require HCF for activity. The neuronal POU IV domain protein, Brn3a, among its many regulatory functions has been described as an enhancer of the NGF receptor trkA, during development in mouse. I therefore investigated the possible link between Brn3a, TrkA, NGF signaling, HCF, Zhangfei and HSV-1 latency and reactivation. I hypothesized that Zhangfei would also suppress the ability of Brn3a to activate the expression of TrkA and that this would have an impact on NGF-TrkA signaling and, consequently on HSV-1 reactivation from latency.<p>My first study determined which Brn3a/trkA promoter interactions were important for trkA transcription. I constructed a plasmid that contains 1043 base pairs of genomic sequences that extend from 30 nucleotides upstream of trkA coding region. In contrast to previous data, a short 190 bp region that lies proximal to the trkA initiation codon was sufficient for Brn3a trans-activation in NGF-differentiated PC12, Vero and human medulloblastoma cells. At least two portions of the 190 bp fragment bind to Brn3a. In addition, Brn3a increased endogenous levels of trkA transcripts in PC12 cells and initiated trkA expression in medulloblastoma cells, which normally do not express trkA. <p>The second step was to determine the effects of HCF and Zhangfei association with Brn3a on trkA trans-activation. I found that Brn3a required HCF for activating the trkA promoter and that Zhangfei has a suppressive effect over Brn3a-trkA activation in non-neuronal cells. In sympathetic neuron-like NGF-treated PC12 cells, Zhangfei did not suppress the ability of Brn3a to activate the TrkA promoter, however, Zhangfei was able capable of inducing the expression of TrkA in the absence of Brn3a. Both Brn3a and Zhangfei induced the expression of endogenous trkA in PC12 cells.<p>Since Vero and PC12 cells are not from human origin I wanted to examine the ability of Zhangfei to induce trkA transcription in medulloblastoma cells, that because of its tumor nature do not express trkA. TrkA transfections in these cells have shown to drive them to cell arrest or apoptosis. Since Zhangfei is not express in medulloblastoma tumors I then used ONS-76 medulloblastoma cells as a model to determine Zhangfeis envolvement in the NGF-trkA signaling pathway.<p> I show herein that in ONS-76 medulloblastoma cells resveratrol, an inducer of apoptosis and differentiation, increased the expression of Zhangfei and trkA as well as Early Growth Response Gene 1 (Egr1), a gene normally activated by NGF-trkA signalling. ONS-76 cells stop growing soon after treatment with resveratrol and a portion of the cell undergo apoptosis. While the induction of Zhangfei in resveratrol-treated cells was modest albeit consistent, the infection of actively growing medulloblastoma cells with an adenovirus vector expressing Zhangfei mimicked the effects of resveratrol. Zhangfei activated the expression of trkA and Egr1 and caused these cells to display markers of apoptosis. The phosphorylation of Erk1, an intermediate kinase in the NGF-trkA signaling critical for differentiation, was observed in Zhangfei infected cells, supporting the hypothesis that Zhangfei is a mediator of trkA-NGF signaling in theses cells leading either to differentiation or apoptosis. Binding of HCF by Zhangfei did not appear to be required for this effect as a mutant of Zhangfei incapable of binding HCF was also able to induce the expression of trkA and Egr1. <p>In in vivo and in vitro models of HSV-1 latency, the virus reactivates when NGF supply to the neuron is interrupted. Based on the above evidence Zhangfei, in HSV-1 latently infected neurons, would have the ability to prolong a state of latency by inducing trkA expression allowing the activation of NGF-trkA signaling pathway. Since NGF is produced by many cell types it is possible that reactivation is triggered not by a decrease in NGF but by a down-regulation of TrkA expression.Therefore, if Zhangfei expression is suppress the trkA signaling could be interrupted or shifted towards apoptosis signaling, this would allow neuronal HCF-binding proteins like Luman, which can activate HSV IE expression, to initiate HSV IE expression and subsequently viral replication.

DISP3 protein, also known as PTCHD2, belongs to the PTCHD family of proteins, which contain a sterol-sensing domain in their structure. The expression of the Disp3 gene is high in neural tissues and is regulated by thyroid hormone. The DISP3 gene is associated with development and progression of certain types of tumors, as well as with development of some neural pathologies. Neural stem cells also display high expression of the Disp3 gene. Neural stem cells are defined by their capability to self-renewal and capacity to differentiate into the basic types of neural cells - neurons, astrocytes, and oligodendrocytes. Precise regulation of the balance between proliferation and differentiation of neural stem cells is crucial for development of the central nervous system and its subsequent proper functioning, and disruption of this balance may lead to development of various pathologies. In this work we mainly focused on describing the function of the DISP3 protein in neural cells and tissues. We have shown that during differentiation of neural stem cells, the expression of the Disp3 gene is significant decreased. Furthermore, we have found that in neural stem and progenitor cells, the increased expression of the Disp3 gene promotes their proliferation. Moreover, when Disp3 expression was disrupted, the...