Stem cell treatments for spinal cord injury (SCI) are effective in pre-clinical animal model research but not yet for humans. Two promising stem cell repair strategies involve (1) endogenous neural stem/progenitor cells (NSPCs) and (2) induced pluripotent stem cells (iPSCs). Delineating species differences in spinal cord NSPC biology is essential to inform human SCI endogenous regeneration and repair. Understanding the phenotypic differences between iPSC-derived NSPCs and primary spinal cord NSPCs would also improve the clinical application of iPSC-derived NSPC therapy in human SCI. To directly compare the molecular and functional attributes of spinal cord NSPCs between humans and animal models of SCI, we designed an in vitro model that allows the characterization of primary human, pig, and rat NSPCs under identical conditions. We found an enrichment of transcription factors in NSPCs of either species that may underlie their differentiation and proliferation potentials. Specifically, human NSPCs are neurogenic, whereas pig and rat NSPCs are gliogenic. Also, the proliferation rate of human and pig NSPCs is less than rat NSPCs. Subsequently, we expanded our in vitro model to examine the responses of NSPCs to inflammation and regenerative factors. Surprisingly, inflammation had induced glial scarring mechanisms from pig and rat NSPCs but potentiated neurogenesis of human NSPCs. We also found species-specific responses to regenerative factors that depend on the type of factor used, concentration, and duration of treatment. To assess the extent that iPSC-derived NSPCs phenocopy primary spinal cord NSPCs, we created iPSC-derived NSPCs with a previously reported brain or spinal cord phenotype and directly compared them with isogenic primary NSPCs. We found that iPSC-derived NSPCs exhibit an earlier developmental stage and a greater proliferation rate. We also found that primary NSPCs possess a unique differentiation potential and regional polarity along the rostral-caudal and dorsoventral axes. In summary, we discovered that species differences in NSPC biology exist between human and animal primary spinal cord NSPCs and that iPSC-derived NSPCs do not recapitulate the transcriptional nor functional attributes of primary spinal cord NSPCs. This thesis highlights the translational gap between pre-clinical research and the clinical application of stem cell treatments that target endogenous NSPCs or transplant iPSC-derived NSPCs.

Adult hippocampal neurogenesis is orchestrated by neural stem cell (NSC) activity. Some associations exist between autophagy and neurogenesis, yet much remains unknown about autophagic regulation of adult neurogenesis. This thesis interrogates the requirement and role of Beclin1 and Atg5, two regulators of autophagy, in the formation of adult hippocampal neurons. To examine adult brain NSCs, the experiments presented in the first objective of this thesis test the ability to isolate adult NSCs using flow cytometry and a DNA-binding dye, DyeCycleViolet. While adult NSCs could not be isolated from the adult neurogenic niches using this methodology, it was effective in isolating endothelial cells. This provided valuable insight on the use of DNA-binding dyes and a new method for isolation of brain endothelial cells. The next objective determines the role of Beclin1 in adult NSCs and their progeny using an inducible model. Beclin1 loss in Nestin-expressing hippocampal NSCs resulted in reduced proliferation, autophagy, and adult neurogenesis within one month. Single-cell RNA sequencing and other methods illuminated that loss of Beclin1 resulted in mitosis reduction, disrupted mitotic regulation of chromatin maintenance, and induction of DNA damage. The final objective first tests whether Beclin1 loss results in similar deficits within GLAST-expressing NSCs and progeny. This model mirrored neurogenesis deficits and requirement of Beclin1 in mitosis and DNA maintenance. Next, to test whether this phenotype occurs with other autophagy proteins, Atg5 was removed from GLAST NSCs. This resulted in reduced autophagy and a transient decrease in neurons in the absence of any effect on NSC proliferation. Thus, proliferation deficits are unique to Beclin1 loss and do not underlie reduced adult hippocampal neurogenesis after Atg5 removal. This work demonstrates a novel discovery of mitosis regulation in adult NSCs by Beclin1, and individual roles of Beclin1 and Atg5 in neurogenesis.

Aging of the brain is the leading risk factor for neurodegenerative diseases and brain cancers and has deleterious effects on brain functions. It follows that attempts to reverse the aging process may be therapeutically valuable. Neural stem cells (NSC) have been shown to play a critical role in maintaining brain functions, and their number is severely decreased with age. The development of senescence-like characteristics and declining functions in NSCs have been proposed to be responsible for brain aging and tumorigenesis. MS-818 is a pyrrolopyrimidine that has been shown to increase the NSC population and reverse the decline of behavioral function in aged rodent models. While MS-818 has demonstrated such benefits, the mechanism by which it affects particular pathways of biological age in NSCs is not yet known. Understanding how MS-818 relates to the molecular mechanisms underlying cellular aging may help accelerate the development of anti-aging therapies for neurodegenerative diseases and cancer. This study attempts to elucidate the mechanism of action of MS-818 on NSCs using an in vitro accelerated-aging model produced by Hydroxyurea (HU) treatment. Our analysis of NSC population size post-MS-818 exposure supports the idea that MS-818 treatment can increase NSC proliferation. qPCR analysis of aging-related genes revealed HU treatment produced a trend of increased p16 and Il-6 and decreased Lamin B1 relative expression, supporting the notion that HU treatment can induce senescence in NSCs. MS-818 treatment alone also produced notable trends for targets including BRCA1. In addition, MS-818 treatment post-HU exposure appeared to influence the relative expression of targets, including PGC1a and Lamin B1. Such MS-818 treatment produced similarly noteworthy trends for the expression of genes including PGC1a, Lamin B1, BRCA1, RPTOR, and Il-6, whether in media containing 2.5% or 7.5% serum. These results indicate that MS-818 may have influenced some aging-related pathways.

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While antiretroviral therapy (ART) regimens have significantly decreased the mortality rate in patients with HIV-1 infection and subsequent opportunistic infections, the co-morbidities continue to rise. Some of these co-morbidities include cardiomyopathies, metabolic dysfunction, accelerated aging, and most notably, neurocognitive deficits. HIV-1 associated neurocognitive disorders (HAND) denote a spectrum of neurocognitive deficits that are either asymptomatic in nature (asymptomatic neurocognitive impairments, ANI), mild to moderate in intensity (mild neurocognitive disorders, MND), or robust in nature (HIV-associated dementia, HAD). Thanks to the development of ART regimens, the incidence of HAD dramatically decreased. However, the emergence of ANI and MND continues to increase in the HIV-1 patient population. While the multifaceted nature behind the central nervous system (CNS) neuropathology of HIV-1 infection is not completely understood, dysregulated blood-brain barrier (BBB) integrity and the “Trojan-Horse” type mechanism of HIV-1 infection have been proposed as the cellular mechanisms underlying HAND. HIV-1 infects CD4+ T-lymphocytes and monocytes in the peripheral circulatory system. After these infected cells cross the BBB into the CNS, they release toxic viral proteins and viral particles onto microglia and astrocytes. These glial cells become activated, and release a plethora of inflammatory cytokines that further damage neurons via dysregulated neurotransmitter homeostasis, synaptodendritic damage, and calcium-mediated apoptotic pathways. At the same time, the virus may establish a state of latency in these microglia, perivascular macrophages, and astrocytes, which would allow for the long-term persistence of HIV-1 in the CNS. Recently, several studies have demonstrated that neural stem cells (NSCs) are capable of being productively and latently infected with HIV-1. This may be due to the fact that the hippocampal subgranular zone (SGZ), the subventricular zone (SVZ), and the circumventricular organs are highly vascularized, allowing potential direct contact of HIV-1 with NSCs. Additionally, the “Trojan” T-cells and macrophages could possibly release viral particles directly onto NSCs, and also transmit the virus through the formation of immunological synapses with NSCs. Therefore, the central hypothesis in this dissertation is that NSCs may serve as a novel CNS reservoir through which HIV-1 infection persists, and subsequently lead to neurocognitive impairments through dysregulating adult neurogenesis. Adult neurogenesis is a dynamic process that describes the generation of new neurons and glial cells from NSCs and neural progenitor cells (NPCs). This process mainly takes place in two areas of the brain: the SVZ around the lateral ventricles, and the SGZ within the dentate gyrus of the hippocampus. New neurons generated in these two neurogenic niches integrate into their respective circuitries to modulate olfactory stimuli and aid in memory acquisition/consolidation processes. Most of previous studies on the role of HIV-1 in neurogenesis focused on single viral proteins rather than the entire integrated proviral genome, and did not correlate these neurogenic deficits to neurobehavioral outcomes. Therefore, the overall objective of the studies proposed in this dissertation is to further validate the feasibility and efficiency of HIV-1 infection in NSCs at both the in vitro and in vivo levels, and explore the correlation of HIV-induced adult neurogenic deficits with neurocognitive dysfunction. The first set of studies utilized an EcoHIV reporter virus to infect mouse NSCs both in vitro and in vivo. This was done because the native HIV-1 virus is incapable of infecting non-human cells, while EcoHIV has been engineered to infect murine cells using the gp80 envelope protein. Our initial studies revealed that EcoHIV preferentially infected NSCs rather than NPCs. Additionally, a 3-day live imaging study revealed that some NSCs were infected at different time points when compared to other cells. This raised credence to the possibility that these infected NSCs/NPCs were generating new viruses which were seeding new infection. NSCs were also capable of propagating higher levels of EcoHIV transcription after treatment with latency reversing agents. Furthermore, EcoHIV infection persisted in a small number of astrocytes during the differentiation process. Subsequent studies assessed whether differentiated neurons and glial cells were vulnerable to EcoHIV infection. Our studies showed that only a small percentage of astrocytes and oligodendrocytes were infected by EcoHIV. Throughout these studies, differentiated neurons were shown to be resistant to HIV-1 infection. These in vitro findings were further validated in vivo. Histological analysis revealed that NSCs were more vulnerable to EcoHIV infection than NPCs. Notably, a small percentage of neuroblasts harbored EcoHIV, though microglia cells were infected at a significantly higher number. Altogether, these findings further solidify NSCs as a novel reservoir through which HIV-1 infection can persist in the CNS. Such findings raised the possibility that HIV-1 in NSCs may dysregulate neurogenesis. The next set of studies in this dissertation elucidated the possible role of HIV-1 infection or viral protein productions in NSCs in regulating adult neurogenesis. Specific parameters analyzed included NSC quiescence, early-stage and middle-stage lineage differentiation, and late-stage neuronal maturation. We performed a series of in vitro and in vivo studies using the HIV-1 Tg26 transgenic mouse model, which mimics HIV-1 patients suffering from low-level and chronic stress from HIV-1 viral proteins in the ART era. NSC culture studies from HIV-1 Tg26 transgenic mice and their wild-type (WT) littermates revealed that Tg26 mouse NSCs were unable to form as many primary neurospheres as WT NSCs. Additionally, when the NSCs were stratified by size, Tg26 NSCs formed lower numbers of smaller-sized primary neurospheres and more larger-sized primary neurospheres. These findings demonstrated that low-level chronic HIV-1 infection robustly reduces the NSC pool, and hampers the initial differentiation process from NSCs to NPCs. In vitro differentiation analyses revealed that compared to WT NSCs, Tg26 NSCs had a lower propensity to differentiate towards a neuronal phenotype, and instead generated more astrocytes. These findings were further confirmed through in vivo hippocampal neural lineage analysis in the SGZs of both WT and Tg26 mice. Subsequent retroviral labeling studies in the SGZ revealed that newborn dentate granule neurons in Tg26 mice had lower dendritic complexity and decreased apical dendritic spine density, when compared to dentate granule neurons from WT mice. These studies further demonstrated that adult neurogenesis is dysregulated upon persistent HIV-1 challenge or infection in NSCs. Further studies sought to examine if HIV-1 Tg26 transgenic mice had any cognitive deficits. We specifically focused on middle-aged WT and Tg26 mice, since the HIV-1 patient population is increasing in age thanks to ART regimens, and thus are more susceptible to cognitive decline than younger HIV-1 patients. We also took into account the factor of biological sex into the behavioral studies. Five types of behavioral assessments revealed sex-specific deficits in Tg26 mice. Specifically, male Tg26 mice exhibited social novelty deficits, and short and long-term spatial memory impairments. On the other hand, female Tg26 mice only manifested spatial learning deficits and short-term spatial memory impairments. Both male and female Tg26 mice had preserved physiological and reflexive functioning, in addition to intact contextual and cued fear conditioning responses. We speculated that these sex-specific differences were due to defects in adult neurogenesis during aging. Through hippocampal neurogenic analysis, we showed that middle-aged male Tg26 mice had an accelerated depletion of the NSC pool and decreased number of neuroblasts. Middle-aged female Tg26 mice have decreased pools of NSCs and NPCs, as well as decreased number of neuroblasts. In conclusion, we have effectively demonstrated that HIV-1 is capable of infecting NSCs at relatively low efficiencies. While differentiated neurons were incapable of sustaining HIV-1 infection, a small percentage of differentiated astrocytes, oligodendrocytes, neuroblasts, and microglia were susceptible to infection. These results led us to investigate the role of dysregulated adult neurogenesis in HIV-1 Tg26 mice, and if this process led to the progression of HAND. Our comprehensive in vitro and in vivo studies demonstrated that HIV-1 induced NSC quiescence, inhibited neuronal differentiation, and promoted astroglial lineage differentiation. Additionally, newborn dentate granule neurons in Tg26 mice had lower dendritic complexity and dendritic spine densities. Finally, both male and female Tg26 mice had varying degrees of cognitive deficits, which was attributed to differing hippocampal neurogenic dynamics during the aging process. Further studies should explore how to restore the neurogenic process during aging in these Tg26 mice. Transcriptomic analysis, such as single cell RNA-sequencing studies, could also possibly assist in further understanding HIV-1 proviral expression changes in differing cellular types along the NSC lineage progression. / Biomedical Sciences

Within the last decade, neurodegenerative diseases such as Alzheimer’s and Parkinson’s have emerged as one of the top 5 leading causes of death globally, and there is currently no cure. All neurodegenerative diseases lead to loss of the functional cells in the nervous system, the neurons. One therapeutic approach is to replace the damaged and lost neurons with new, healthy neurons. Unfortunately, this is a difficult endeavor since mature neurons are not capable of cell division. Instead, researchers are turning to neural stem cells, which are able to self-renew and be rapidly expanded before being differentiated into functional cell phenotypes, such as neurons, allowing for large numbers of cells to be generated in vitro. Controlled differentiation of human neural stem cells into new neurons has been of interest due to the immense potential for improving clinical outcomes. Adult neural stem cell behavior, however, is not well understood and the transplanted stem cells are at risk for tumorigenesis. The focus of this dissertation is the development of engineered biomaterials as tools to study human neural stem cell behavior and neurogenesis (differentiation). A novel cell penetrating peptide was developed to enhance intracellular delivery of retinoic acid, a bioactive lipid known to induce differentiation. A hydrogel platform fabricated from hyaluronic acid, a naturally-occurring polysaccharide found in brain extracellular space, was designed to serve as a biomimetic soft substrate with similar mechanical properties to the brain. The biological behavior of the stem cells was characterized in response to chemical and physical cues. / Bioengineering

In this work, a hydrogel made from hyaluronic acid is synthesized and utilized to study neural stem cell behavior within a custom tailored three dimensional microenvironment. The physical properties of the hydrogel have been optimized to create an environment conducive for neural stem cell differentiation by mimicking the native brain extracellular matrix (ECM) environment. The physical properties characterized include degree of methacrylation, swelling ratios, enzymatic degradation rates, and viscoelastic moduli. One dimensional proton nuclear magnetic resonance (1HNMR) confirms modification of the hyaluronic acid polymers, and is used to quantify the degree of methacrylation. Rheological measurements are made to quantify the viscoelastic moduli. Further post-processing by lyophilization leads to generation of large voids to facilitate re-swelling and cell infiltration. ReNcell VM (RVM), and adult human neural stem cell line derived from the ventral mesencephalon, are cultured and differentiated inside the hydrogel for up to 2 weeks. Differentiation is characterized by immunocytochemistry (ICC) and real time quantitative polymerase chain reaction (qRT-PCR). / Bioengineering

Traumatic brain injury (TBI) is a major cause of disability worldwide. No effective treatment is currently available to restore function to the injured brain. After injury, massive neuronal death occurs which can result in long-lasting cognitive dysfunction. Following immediate mechanical damage, a series of secondary effects of injury occur including evolving neuronal damage, inflammation, astrocyte reactivation, blood brain barrier disruption and other physiological effects. Additionally, neural stem cell (NSC) proliferation has been observed following TBI, suggestive of an endogenous attempt to repair the brain. Stimulating proliferation of NSCs is a promising strategy to facilitate recovery following TBI, but the mechanisms underlying NSC proliferation remain unknown. In this work, we have addressed the following specific aims. In the first aim, we determined the role of Shh signaling in NSC proliferation after TBI. Using a fluid percussion model of TBI and conditional transgenic animal models, we investigated the role astrocytes play in NSC proliferation. Using a Sonic hedgehog (Shh) pathway inhibitor, we found that NSC proliferation after TBI relies on Shh signaling. In the second aim, we determined the role of astrocyte activation in NSC proliferation after TBI. Using transgenic tools, we determined that astrocytes are a major cellular source of Shh and that astrocyte-specific deletion of Shh inhibited NSC proliferation. This indicates that NSC proliferation relies on Shh signaling and that astrocytes represent the key cellular source. In the final aim, we sought to define the functional requirement of Nestin in NSC proliferation. Recent studies in our lab found that Nestin, an intermediate filament protein predominantly expressed by NSCs, played a role in Shh signaling in the setting of medulloblastoma cells. Here, we found that knockdown of Nestin impaired Shh signal transduction and Shh-driven proliferation in NSCs. Further, we generated a new mouse model allowing conditional deletion of Nestin in NSCs to determine whether Nestin played a similar role a non-neoplastic setting. Conditional deletion of Nestin in NSCs abolished the proliferation of hippocampal NSCs after TBI. These findings reveal the critical role of Nestin in Shh signaling and proliferation in NSCs following TBI. Our studies elucidate the cellular and molecular basis for NSC proliferation after TBI, which pave the road for development of therapeutic approaches to treat TBI by augmenting endogenous NSC regeneration. / Cancer Biology & Genetics