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.

The RNA-binding protein Quaking (QKI) plays a fundamental role in post-transcriptional gene regulation during mammalian nervous system development. QKI is well known for advancing oligodendroglia differentiation and myelination, however, its functions in astrocytes and embryonic central nervous system (CNS) development remain poorly understood. Uncovering the complete spectrum of QKI molecular and functional repertoire is of additional importance in light of growing evidence linking QKI dysfunction with human disease, including schizophrenia and glioma. This thesis summarizes my contribution to fill this gap of knowledge.         In a first attempt to identify the QKI-mediated molecular pathways in astroglia, we studied the effects of QKI depletion on global gene expression in the human astrocytoma cell line. This work revealed a previously unknown role of QKI in regulating immune-related pathways. In particular, we identified several putative mRNA targets of QKI involved in interferon signaling, with possible implications in innate cellular antiviral defense, as well as tumor suppression. We next extended these investigations to human primary astrocytes, in order to more accurately model normal brain astrocytes. One of the most interesting outcomes of this analysis was that QKI regulates expression of transcripts encoding the Glial Fibrillary Acidic Protein, an intermediate filament protein that mediates diverse biological functions of astrocytes and is implicated in numerous CNS pathologies. We also characterized QKI splice variant composition and subcellular expression of encoded protein isoforms in human astrocytes. Finally, we explored the potential use of zebrafish as a model system to study neurodevelopmental functions of QKI in vivo. Two zebrafish orthologs, qkib and qki2, were identified and found to be widely expressed in the CNS neural progenitor cell domains. Furthermore, we showed that a knockdown of qkib perturbs the development of both neuronal and glial populations, and propose neural progenitor dysfunction as the primary cause of the observed phenotypes.        To conclude, the work presented in this thesis provides the first insight into understanding the functional significance of the human QKI in astroglia, and introduces zebrafish as a novel tool with which to further investigate the importance of this gene in neural development.

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.

abstract: Traumatic brain injury (TBI) is a significant public health concern in the U.S., where approximately 1.7 million Americans sustain a TBI annually, an estimated 52,000 of which lead to death. Almost half (43%) of all TBI patients report experiencing long-term cognitive and/or motor dysfunction. These long-term deficits are largely due to the expansive biochemical injury that underlies the mechanical injury traditionally associated with TBI. Despite this, there are currently no clinically available therapies that directly address these underlying pathologies. Preclinical studies have looked at stem cell transplantation as a means to mitigate the effects of the biochemical injury with moderate success; however, transplants suffer very low retention and engraftment rates (2-4%). Therefore, transplants need better tools to dynamically respond to the injury microenvironment. One approach to develop new tools for stem cell transplants may be to look towards the endogenous repair response for inspiration. Specifically, activated cell types surrounding the injury secrete the chemokine stromal cell-derived factor-1α (SDF-1α), which has been shown to play a critical role in recruiting endogenous neural progenitor/stem cells (NPSCs) to the site of injury. Therefore, it was hypothesized that improving NPSC response to SDF-1α may be a viable mechanism for improving NPSC transplant retention and migration into the surrounding host tissue. To this end, work presented here has 1. identified critical extracellular signals that mediate the NPSC response to SDF-1α, 2. incorporated these findings into the development of a transplantation platform that increases NPSC responsiveness to SDF-1α and 3. observed increased NPSC responsiveness to local exogenous SDF-1α signaling following transplantation within our novel system. Future work will include studies investigating NSPC response to endogenous, injury-induced SDF-1α and the application of this work to understanding differences between stem cell sources and their implications in cell therapies. / Dissertation/Thesis / Doctoral Dissertation Bioengineering 2015

abstract: Several debilitating neurological disorders, such as Alzheimer's disease, stroke, and spinal cord injury, are characterized by the damage or loss of neuronal cell types in the central nervous system (CNS). Human neural progenitor cells (hNPCs) derived from human pluripotent stem cells (hPSCs) can proliferate extensively and differentiate into the various neuronal subtypes and supporting cells that comprise the CNS. As such, hNPCs have tremendous potential for disease modeling, drug screening, and regenerative medicine applications. However, the use hNPCs for the study and treatment of neurological diseases requires the development of defined, robust, and scalable methods for their expansion and neuronal differentiation. To that end a rational design process was used to develop a vitronectin-derived peptide (VDP)-based substrate to support the growth and neuronal differentiation of hNPCs in conventional two-dimensional (2-D) culture and large-scale microcarrier (MC)-based suspension culture. Compared to hNPCs cultured on ECMP-based substrates, hNPCs grown on VDP-coated surfaces displayed similar morphologies, growth rates, and high expression levels of hNPC multipotency markers. Furthermore, VDP surfaces supported the directed differentiation of hNPCs to neurons at similar levels to cells differentiated on ECMP substrates. Here it has been demonstrated that VDP is a robust growth and differentiation matrix, as demonstrated by its ability to support the expansions and neuronal differentiation of hNPCs derived from three hESC (H9, HUES9, and HSF4) and one hiPSC (RiPSC) cell lines. Finally, it has been shown that VDP allows for the expansion or neuronal differentiation of hNPCs to quantities (>1010) necessary for drug screening or regenerative medicine purposes. In the future, the use of VDP as a defined culture substrate will significantly advance the clinical application of hNPCs and their derivatives as it will enable the large-scale expansion and neuronal differentiation of hNPCs in quantities necessary for disease modeling, drug screening, and regenerative medicine applications. / Dissertation/Thesis / Masters Thesis Bioengineering 2016

abstract: Neurodegenerative diseases such as Alzheimer’s Disease, Parkinson’s Disease and Amyotrophic Lateral Sclerosis are marked by the loss of different types of neurons and glial cells in the central nervous system (CNS). Human Pluripotent Stem Cell (hPSC)-derived Neural Progenitor Cells (hNPCs) have the ability to self-renew indefinitely and to differentiate into various cell types of the CNS. HNPCs can be used in cell based therapies and have the potential to reverse or arrest neurodegeneration and to replace lost neurons and glial cells. However, the lack of completely defined, scalable systems to culture these cells, limits their therapeutic and clinical applications. In a previous study, a completely defined, robust, synthetic peptide- a Vitronectin Derived Peptide (VDP) that supports the long term expansion and differentiation of various embryonic and induced pluripotent stem cell (hESC/hIPSC) derived hNPC lines on two dimensional (2D) tissue culture plates was identified. In this study, the culture of hNPCs was scaled up using VDP coated microcarriers (MC). VDP MC were able to support the long term expansion of hESC and hiPSC derived hNPCs over multiple passages and supported higher fold changes in cell densities, compared to VDP coated 2D surfaces. VDP MC also showed the ability to support the neuronal differentiation of hNPCs, and produced mature neurons expressing several neuronal, neurotransmitter and cortical markers. Additionally, alzheimer’s disease (AD) relevant phenotypes were studied in patient hIPSC derived hNPCs cultured on laminin MC to assess if the MC culture system could be used for disease modelling and drug screening. Finally, a microcarrier based bioreactor system was developed for the large scale expansion of hNPCs, exhibiting more than a five-fold change in cell density and supporting more than 100 million hNPCs in culture. Thus, the development of a xeno-free, scalable system allows hNPC culture under standard and reproducible conditions in quantities required for therapeutic and clinical applications. / Dissertation/Thesis / Masters Thesis Bioengineering 2017

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.