Whole document restricted, see Access Instructions file below for details of how to access the print copy. / Huntington’s disease [HD] is a debilitating adult onset inherited neurodegenerative disorder with primary degeneration in the striatum and widespread secondary degeneration throughout the brain. There are currently no clinical treatments to prevent onset, delay progression or replace lost neurons. Striatal cell transplantation strategies under clinical evaluation appear viable and effective for the treatment of HD. However, the future of regenerative medicine lies in developing renewable, expandable multipotent neural cell sources for transplantation. This Thesis has investigated a range of novel developments for enhancing the therapeutic potential of neural progenitor cell transplantation in a quinolinic acid [QA] lesion rat model of HD using two cell sources, adult neural progenitor cells and human embryonic stem cell [hESC] derived neural progenitor cells. Chapter Three identified a novel method for in vitro lithium priming of adult neural progenitor cells which enhances their neurogenic potential at the expense of glial formation. Chapter Four demonstrated that lithium priming of adult neural progenitor cells altered their phenotypic fate in vivo after transplantation, enhancing regional specific differentiation and efferent projection formation. The therapeutic potential of this strategy was demonstrated by accelerated acquisition of motor function benefits in the QA model. Chapter Five then demonstrated the ability for post transplantation environmental enrichment to modify therapeutic functional outcomes in the QA lesion model, and through lithium priming and enrichment demonstrated that adult neural progenitors are amenable to combinatorial interventions which can alter their phenotypic fate and enhance anatomical integration. Chapter Six investigated the in vivo effects of in vitro noggin priming of hESC derived neural progenitor cells and identified enhanced safety and neuronal differentiation in the QA lesioned striatum after noggin priming. Furthermore Chapter Seven provided evidence for functional reconstruction and therapeutic functional benefits from transplantation of noggin primed hESC derived neural progenitor cells and also highlighted the need for systematic evaluations of hESC derived transplants to optimise their safety in vivo. These results are beneficial in demonstrating the realistic therapeutic potential held by these two cell sources. They demonstrate how transient interventions can enhance therapeutic outcomes of neural progenitor cell transplantation for HD and have developed the understanding of neural progenitor cell transplantation as a therapeutic tool, bringing transplantation from different cell sources closer to eventual translation for HD sufferers.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / Huntington’s disease [HD] is a debilitating adult onset inherited neurodegenerative disorder with primary degeneration in the striatum and widespread secondary degeneration throughout the brain. There are currently no clinical treatments to prevent onset, delay progression or replace lost neurons. Striatal cell transplantation strategies under clinical evaluation appear viable and effective for the treatment of HD. However, the future of regenerative medicine lies in developing renewable, expandable multipotent neural cell sources for transplantation. This Thesis has investigated a range of novel developments for enhancing the therapeutic potential of neural progenitor cell transplantation in a quinolinic acid [QA] lesion rat model of HD using two cell sources, adult neural progenitor cells and human embryonic stem cell [hESC] derived neural progenitor cells. Chapter Three identified a novel method for in vitro lithium priming of adult neural progenitor cells which enhances their neurogenic potential at the expense of glial formation. Chapter Four demonstrated that lithium priming of adult neural progenitor cells altered their phenotypic fate in vivo after transplantation, enhancing regional specific differentiation and efferent projection formation. The therapeutic potential of this strategy was demonstrated by accelerated acquisition of motor function benefits in the QA model. Chapter Five then demonstrated the ability for post transplantation environmental enrichment to modify therapeutic functional outcomes in the QA lesion model, and through lithium priming and enrichment demonstrated that adult neural progenitors are amenable to combinatorial interventions which can alter their phenotypic fate and enhance anatomical integration. Chapter Six investigated the in vivo effects of in vitro noggin priming of hESC derived neural progenitor cells and identified enhanced safety and neuronal differentiation in the QA lesioned striatum after noggin priming. Furthermore Chapter Seven provided evidence for functional reconstruction and therapeutic functional benefits from transplantation of noggin primed hESC derived neural progenitor cells and also highlighted the need for systematic evaluations of hESC derived transplants to optimise their safety in vivo. These results are beneficial in demonstrating the realistic therapeutic potential held by these two cell sources. They demonstrate how transient interventions can enhance therapeutic outcomes of neural progenitor cell transplantation for HD and have developed the understanding of neural progenitor cell transplantation as a therapeutic tool, bringing transplantation from different cell sources closer to eventual translation for HD sufferers.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / Huntington’s disease [HD] is a debilitating adult onset inherited neurodegenerative disorder with primary degeneration in the striatum and widespread secondary degeneration throughout the brain. There are currently no clinical treatments to prevent onset, delay progression or replace lost neurons. Striatal cell transplantation strategies under clinical evaluation appear viable and effective for the treatment of HD. However, the future of regenerative medicine lies in developing renewable, expandable multipotent neural cell sources for transplantation. This Thesis has investigated a range of novel developments for enhancing the therapeutic potential of neural progenitor cell transplantation in a quinolinic acid [QA] lesion rat model of HD using two cell sources, adult neural progenitor cells and human embryonic stem cell [hESC] derived neural progenitor cells. Chapter Three identified a novel method for in vitro lithium priming of adult neural progenitor cells which enhances their neurogenic potential at the expense of glial formation. Chapter Four demonstrated that lithium priming of adult neural progenitor cells altered their phenotypic fate in vivo after transplantation, enhancing regional specific differentiation and efferent projection formation. The therapeutic potential of this strategy was demonstrated by accelerated acquisition of motor function benefits in the QA model. Chapter Five then demonstrated the ability for post transplantation environmental enrichment to modify therapeutic functional outcomes in the QA lesion model, and through lithium priming and enrichment demonstrated that adult neural progenitors are amenable to combinatorial interventions which can alter their phenotypic fate and enhance anatomical integration. Chapter Six investigated the in vivo effects of in vitro noggin priming of hESC derived neural progenitor cells and identified enhanced safety and neuronal differentiation in the QA lesioned striatum after noggin priming. Furthermore Chapter Seven provided evidence for functional reconstruction and therapeutic functional benefits from transplantation of noggin primed hESC derived neural progenitor cells and also highlighted the need for systematic evaluations of hESC derived transplants to optimise their safety in vivo. These results are beneficial in demonstrating the realistic therapeutic potential held by these two cell sources. They demonstrate how transient interventions can enhance therapeutic outcomes of neural progenitor cell transplantation for HD and have developed the understanding of neural progenitor cell transplantation as a therapeutic tool, bringing transplantation from different cell sources closer to eventual translation for HD sufferers.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / Huntington’s disease [HD] is a debilitating adult onset inherited neurodegenerative disorder with primary degeneration in the striatum and widespread secondary degeneration throughout the brain. There are currently no clinical treatments to prevent onset, delay progression or replace lost neurons. Striatal cell transplantation strategies under clinical evaluation appear viable and effective for the treatment of HD. However, the future of regenerative medicine lies in developing renewable, expandable multipotent neural cell sources for transplantation. This Thesis has investigated a range of novel developments for enhancing the therapeutic potential of neural progenitor cell transplantation in a quinolinic acid [QA] lesion rat model of HD using two cell sources, adult neural progenitor cells and human embryonic stem cell [hESC] derived neural progenitor cells. Chapter Three identified a novel method for in vitro lithium priming of adult neural progenitor cells which enhances their neurogenic potential at the expense of glial formation. Chapter Four demonstrated that lithium priming of adult neural progenitor cells altered their phenotypic fate in vivo after transplantation, enhancing regional specific differentiation and efferent projection formation. The therapeutic potential of this strategy was demonstrated by accelerated acquisition of motor function benefits in the QA model. Chapter Five then demonstrated the ability for post transplantation environmental enrichment to modify therapeutic functional outcomes in the QA lesion model, and through lithium priming and enrichment demonstrated that adult neural progenitors are amenable to combinatorial interventions which can alter their phenotypic fate and enhance anatomical integration. Chapter Six investigated the in vivo effects of in vitro noggin priming of hESC derived neural progenitor cells and identified enhanced safety and neuronal differentiation in the QA lesioned striatum after noggin priming. Furthermore Chapter Seven provided evidence for functional reconstruction and therapeutic functional benefits from transplantation of noggin primed hESC derived neural progenitor cells and also highlighted the need for systematic evaluations of hESC derived transplants to optimise their safety in vivo. These results are beneficial in demonstrating the realistic therapeutic potential held by these two cell sources. They demonstrate how transient interventions can enhance therapeutic outcomes of neural progenitor cell transplantation for HD and have developed the understanding of neural progenitor cell transplantation as a therapeutic tool, bringing transplantation from different cell sources closer to eventual translation for HD sufferers.

Alzheimer's disease (AD) leads to neurodegeneration resulting in cognitive and physical impairments. AD is denoted by accumulation of intracellular neurofibrillary tangles, known as tau, and extracellular plaques of the amyloid beta protein (Aβ). Aβ results from the proteolytic cleavage of the amyloid precursor protein (APP) by β- and gamma-secretases in the amyloidogenic pathway. Although, Aβ has been widely studied for neurodegeneration, the role of APP in both, the healthy and diseased conditions, has not yet been entirely understood. The function that APP has in neural stem cell (NSC) proliferation, differentiation, and migration during adult neurogenesis has been previously studied. Additionally, APP has be shown to be overexpressed after neural damage resulted from conditions, such as AD and traumatic brain injury (TBI). In this study, the role of APP in in vitro damaged neural tissue cells was further investigated by evaluating neural progenitor cell proliferation, migration, and differentiation after a scratch assay. For these purposes, induced pluripotent stem (iPS) cells from AD patients were differentiated into neural progenitor cells to model the disease conditions and later treated with Phenserine to reduce their levels of APP expression. The results suggested that APP may enhance neural progenitor cell proliferation and glial differentiation while inhibiting neural progenitor cell migration and neuronal cell specialization after neural tissue damage.

Human immunodeficiency virus type 1 (HIV-1) is known to cause a spectrum of neurological, behavioral and motor deficits collectively termed as HIV-1 associated neurocognitive impairments (HAND). Opiates augment HIV-related CNS complications through both direct and indirect mechanisms that disrupt glial and neuronal function. All CNS macroglia and neurons derive from neural progenitor cells (NPCs) during development, and NPCs in the adult brain contribute to repair processes. Since disruptions in NPC function are known to impact CNS populations and brain function in a number of disease/injury conditions, we determined whether HIV ± opiate exposure affected the maturation and fate of human NPCs (hNPCs). As hNPC infection by HIV has occasionally been reported, we also reexamined this question, and parsed between effects due directly to hNPC infection by HIV, or to hNPC dysfunction caused by the infective milieu. Multiple approaches confirmed the infection of hNPCs by R5 tropic (CCR5 utilizing) HIVBaL, and demonstrated that active infection could be sequentially transferred to naïve hNPCs. Exposure to supernatant from HIVBaL-infected cells (HIVsup) reduced hNPC proliferation and led to premature differentiation into astrocytes and neurons. Morphine co-exposure prolonged hNPC infection and exacerbated functional effects of HIVsup. Neither purified virions nor UV-inactivated HIVsup altered proliferation, indicating that this effect did not require infection. Gene array analysis and RT-qPCR with immunoblot validation suggested that Cdk5 signaling was involved in HIV-morphine interactions. siRNA-mediated knockdown of Cdk5 expression attenuated the effect of HIV-1 and morphine on hNPC proliferation and MAP2 differentiation, but also increased hNPC death. Furthermore, in an attempt to understand the role of mu-opioid receptor (MOR) splice variants on the interactive effect of HIV-1 and morphine on hNPCs, we found that both MOR-1 and MOR-1K are differentially regulated by HIV-1 in these cells. This suggests that these splice variants may have differential actions in the response of hNPCs to HIV-1 and morphine co-exposure. Given the overlap of Cdk5 and MOR signaling, it is likely that MOR-1K and/or MOR-1 converge with Cdk5 in the mechanism underlying HIV-1 and morphine interaction in hNPCs. Overall, dysregulation of hNPC functions by the infectious environment may create cell population imbalances that contribute to CNS deficits in both adult and pediatric patients. Additionally, infected hNPCs may pass virus to their progeny, and serve as an unappreciated viral reservoir. The recent epidemic of opiate/heroin abuse highlights the clinical importance of HIV and opiate interactions.

Neonatal hydrocephalus is a common neurological disorder leading to expansion of the cerebral ventricles. This disease is associated with significant morbidity and mortality and is often fatal if left untreated. Hydrocephalus was first described over 2500 years ago by Hippocrates, the father of medicine, and remains poorly understood today. Current therapies still rely on invasive procedures developed over 60 years ago that are associated with high failure and complication rates. Thus, the identification of molecular mechanisms and the development of non-invasive medical treatments for neonatal hydrocephalus are high priorities for the medical and scientific communities. The prevailing doctrine in the field is that hydrocephalus is strictly a "plumbing problem" caused by impaired cerebrospinal fluid (CSF) flow. Recently, animal models with impaired cilia have provided insight into the mechanisms involved in communicating (non-obstructive) hydrocephalus. However, as a result of a poor understanding of hydrocephalus, no animal studies to date have identified an effective non-invasive treatment. The goal of this thesis project is to investigate the molecular mechanisms underlying this disease and to identify a non-invasive, highly effective treatment strategy. In Chapter 2, we utilize a novel animal model with idiopathic hydrocephalus, mimicking the human ciliopathy Bardet-Biedl Syndrome (BBS), to examine the role of cilia in hydrocephalus. We find that these mice develop communicating hydrocephalus prior to the development of ependymal "motile" cilia, suggesting that this phenotype develops as a result of dysfunctional "primary" cilia. Primary cilia are non-motile and play a role in cellular signaling. These results challenge the current dogma that dysfunctional motile cilia underlies neonatal hydrocephalus and implicate a novel role for primary cilia and cellular signaling in this disease. Chapter 3 focuses on identifying the link between primary cilia and neonatal hydrocephalus. In this chapter, we report that disrupting the molecular machinery within primary cilia leads to faulty PDGFRα signaling and the loss of a particular class of neural progenitor cells called oligodendrocyte precursor cells (OPCs). We find that the loss of OPCs leads to neonatal hydrocephalus. Importantly, we identify the molecular mechanism underlying both the loss of OPCs and the pathogenesis of neonatal hydrocephalus. Chapter 4 explores the therapeutic potential of targeting the defective cellular signaling pathways to treat neonatal hydrocephalus. By targeting the faulty signaling, we restore normal development of oligodendrocyte precursor cells, and curtail the development of hydrocephalus. This work challenges the predominant view of hydrocephalus being strictly a "plumbing problem" treatable solely by surgical diversion of CSF. Here, we propose that hydrocephalus is a neurodevelopmental disorder that can be ameliorated by non-invasive means. Importantly, we introduce novel molecular targets and a non-invasive treatment strategy for this devastating disorder. To our knowledge, we are the first to successfully treat neonatal hydrocephalus in any model organism by targeting neural progenitor cells.

Large-scale expansion of lineage-committed stem cells can provide an excellent ex vivo model for studying complex molecular pathways governing cell fate choices. Also, such cells could be useful for implementing cell therapeutic approaches for treatment of specific disorders involving extensive cellular damage within that lineage. Using growth factors, pluri- and multipotent stem cells have been successfully isolated and cultured from pre- and peri-implantation stage embryos, including trophectoderm, primitive ectoderm, epiblast and primitive endoderm. However, ex vivo expansion of lineage restricted cells from later embryonic lineages and adult tissues have been a challenge. N-myc is a well-characterized member of myc gene family that is known to be essential for the proliferation of numerous progenitor cell types during normal embryonic development of diverse organs including lungs, liver, heart, kidneys and brain. Considering this important role of N-myc, we hypothesized that its regulated activation in these progenitors might allow their expansion in culture. To test this hypothesis, we generated a novel doxycycline-inducible transgenic mouse line that expresses N-myc uniformly across all tissues. Using cortical precursors derived from mid-gestation embryos of these mice, we show that upon doxycycline induced N-myc expression, we can achieve at least a million-fold expansion of multipotent neural precursors within a short span of time in culture. When doxycycline is withdrawn, N-myc expression is turned off and the cells differentiate into neurons and glia. An extensive characterization of the expanded cells revealed that the cells retained their differentiation potential, genomic stability and commitment specific to their origin. The tetracycline-inducible N-myc expressing mouse line might also serve as a source for establishing other than neural lineage committed progenitor cell lines where N-myc has a known role in regulating cell proliferation and differentiation decisions.

Large-scale expansion of lineage-committed stem cells can provide an excellent ex vivo model for studying complex molecular pathways governing cell fate choices. Also, such cells could be useful for implementing cell therapeutic approaches for treatment of specific disorders involving extensive cellular damage within that lineage. Using growth factors, pluri- and multipotent stem cells have been successfully isolated and cultured from pre- and peri-implantation stage embryos, including trophectoderm, primitive ectoderm, epiblast and primitive endoderm. However, ex vivo expansion of lineage restricted cells from later embryonic lineages and adult tissues have been a challenge. N-myc is a well-characterized member of myc gene family that is known to be essential for the proliferation of numerous progenitor cell types during normal embryonic development of diverse organs including lungs, liver, heart, kidneys and brain. Considering this important role of N-myc, we hypothesized that its regulated activation in these progenitors might allow their expansion in culture. To test this hypothesis, we generated a novel doxycycline-inducible transgenic mouse line that expresses N-myc uniformly across all tissues. Using cortical precursors derived from mid-gestation embryos of these mice, we show that upon doxycycline induced N-myc expression, we can achieve at least a million-fold expansion of multipotent neural precursors within a short span of time in culture. When doxycycline is withdrawn, N-myc expression is turned off and the cells differentiate into neurons and glia. An extensive characterization of the expanded cells revealed that the cells retained their differentiation potential, genomic stability and commitment specific to their origin. The tetracycline-inducible N-myc expressing mouse line might also serve as a source for establishing other than neural lineage committed progenitor cell lines where N-myc has a known role in regulating cell proliferation and differentiation decisions.

During spinal cord injury (SCI), glutamate excitotoxicity and astrocytic scar formation can impede repair. In a preliminary study we found that ketamine, a N-methyl-D-aspartate (NMDA) receptor non-competitive antagonist, can contribute to functional recovery post SCI. Therefore, we investigated the cellular basis for this recovery with respect to neural progenitor cells using an in vitro cell culture model. We examined whether ketamine and glutamate influenced the proliferation, differentiation, and migration of differentiating endogenous neural progenitor cells (NPCs) found in the subventricular zone and spinal cord. Our study illustrates that the post functional recovery may have been due to ketamine’s influence on delaying spinal cord NPCs derived astrocyte maturation and migration while increasing radial glial cell migration. These results are promising since ketamine administration may help alleviate some of the adverse affects glutamate has on the NPCs found in the spinal cord following SCI.