Williams Beuren syndrome Syndrome (WBS) and 7q11.23 Duplication Syndrome (Dup7) are rare neurodevelopmental disorders associated with a range of cognitive and behavioural symptoms, caused by the deletion and duplication, respectively, of 26 genes on human chromosome 7q11.23. I have studied the effects of deletion or duplication of two candidate genes, GTF2I and GTF2IRD1, on neural stem cell growth and neurogenesis using cultured primary neuronal precursors from mouse models with gene copy number changes. I found that the number of neuronal precursors and committed neurons was directly related to the copy number of these genes in the mid-gestation embryonic cortex. I further found that in late-gestation embryos, cortical thickness was altered in a similar gene dose-dependent manner, in combination with layer-specific changes in neuronal density. I hypothesize that some of the neurological features of WS and Dup7 stem from these impairments in early cortical development.

Williams Beuren syndrome Syndrome (WBS) and 7q11.23 Duplication Syndrome (Dup7) are rare neurodevelopmental disorders associated with a range of cognitive and behavioural symptoms, caused by the deletion and duplication, respectively, of 26 genes on human chromosome 7q11.23. I have studied the effects of deletion or duplication of two candidate genes, GTF2I and GTF2IRD1, on neural stem cell growth and neurogenesis using cultured primary neuronal precursors from mouse models with gene copy number changes. I found that the number of neuronal precursors and committed neurons was directly related to the copy number of these genes in the mid-gestation embryonic cortex. I further found that in late-gestation embryos, cortical thickness was altered in a similar gene dose-dependent manner, in combination with layer-specific changes in neuronal density. I hypothesize that some of the neurological features of WS and Dup7 stem from these impairments in early cortical development.

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive neuromuscular disease characterized by progressive muscle-wasting and loss of mobility. One-third of patients with DMD are also affected by cognitive impairments such as a lower than average IQ and impaired working memory, comorbid with neuropsychiatric disorders such as anxiety and autism-related behaviours. DMD is caused by mutations in the DMD gene resulting in the deletion of the full-length dystrophin protein (Dp427) and, dependent on mutation, other dystrophin isoforms. These isoforms are predominantly found in the brain and deletion may impact on cognition. The most commonly used animal model to study DMD is the mdx mouse which completely lacks Dp427 but no other DMD isoforms. Although the muscle phenotype is well-established, behavioural characterization of the mdx mouse model has been inconclusive. In this thesis I investigated the hippocampal and amygdala cellular and behavioural phenotypes of the mdx mouse. I show that post-natal neural stem-like cell division in the SGZ is altered in the absence of Dp427 resulting in enhanced symmetric division. I show in vitro that primary mdx cultures are fewer and smaller than wild-type, consistent with an increase in symmetrical self-renewal whereas secondary cultures are fewer and larger, consistent with a shift in symmetric division producing transit-amplifying type 2a daughter cells. I next characterized the mdx mouse model using a battery of behavioural tests. Data presented here show that mdx mice do not exhibit an anxious phenotype, do not display autism-related behaviours, and do not display impairments in and spatial learning or memory. However, associative learning, as measured in the fear conditioning paradigm is enhanced in mdx mice. Lastly, I attempted to generate three different brain-specific dystrophin knock-out mouse models to examine role of other dystrophin isoforms. While none of the models were able to deplete dystrophin from brain, given the inverse relationship between Cre-mediated efficiency and the genetic distance of the loxP sites in the fDMDH mouse employed, I do provide important insight into the presence and absence of the muscle-specific enhancers in constructs commonly used to generate brain-specific mouse models. Taken together, this thesis provides converging evidence to indicate that loss of Dp427 impacts on fear associative learning and stem-cell like division in the SGZ but likely does not underlie the non-progressive cognitive impairments affecting one-third of all DMD patients.

<p>Stem cells have the capacity for both self renewal, and to form all cell types in the body. Interestingly, so called neural stem cells (NSCs) are found in the adult human brain, which is of significance both out of a developmental perspective and from a clinical point of view. At the present moment, the regulation of neural stem cell (NSC) proliferation and fate determination is not completely understood.</p><p>The overall aim of this thesis was to study the mechanisms that regulate NSC proliferation and fate determination <i>in vitro</i> and <i>in vivo</i>. In particular, the roles of the female sex hormone estrogen and the testosterone analogue nandrolone, as well as the melanocortin α-melanocyte stimulating hormone (α-MSH), were analyzed in this context. Also, the breast cancer susceptibility gene one (BRCA-1), was studied in the brain with emphasis on regions containing NSCs.</p><p>Our findings show that estrogen and nandrolone have similar effects on NSCs; both decreased NSC proliferation and increased neurogenesis. Estrogen's ability to reduce proliferation was due to increased levels of p21, an inhibitor of cyclin dependent kinases. In contrast, no change in p21 was observed in the case of nandrolone, indicating differential regulation. Adult rats subjected to nandrolone injections had 30% reduced NSC proliferation in the dentate gyrus, indicating profound effects on NSCs <i>in vivo</i>.</p><p>The melanocortin α-MSH acted as a mitogen by increasing levels of cyclinD1 and retinoblastoma protein; as a result NSC proliferation was doubled.</p><p>Finally, BRCA-1 is expressed while NSCs proliferate, but is drastically down regulated upon differentiation, indicating that BRCA-1 could be used as a possible NSC marker.</p><p>In summary, in this thesis estrogen and nandrolone were identified as NSC regulators which decrease proliferation and positively influence neurogenesis. Also, we have identified the hormone α-MSH as a NSC mitogen, and BRCA-1 as a possible NSC marker.</p>

Stem cells have the capacity for both self renewal, and to form all cell types in the body. Interestingly, so called neural stem cells (NSCs) are found in the adult human brain, which is of significance both out of a developmental perspective and from a clinical point of view. At the present moment, the regulation of neural stem cell (NSC) proliferation and fate determination is not completely understood. The overall aim of this thesis was to study the mechanisms that regulate NSC proliferation and fate determination in vitro and in vivo. In particular, the roles of the female sex hormone estrogen and the testosterone analogue nandrolone, as well as the melanocortin α-melanocyte stimulating hormone (α-MSH), were analyzed in this context. Also, the breast cancer susceptibility gene one (BRCA-1), was studied in the brain with emphasis on regions containing NSCs. Our findings show that estrogen and nandrolone have similar effects on NSCs; both decreased NSC proliferation and increased neurogenesis. Estrogen's ability to reduce proliferation was due to increased levels of p21, an inhibitor of cyclin dependent kinases. In contrast, no change in p21 was observed in the case of nandrolone, indicating differential regulation. Adult rats subjected to nandrolone injections had 30% reduced NSC proliferation in the dentate gyrus, indicating profound effects on NSCs in vivo. The melanocortin α-MSH acted as a mitogen by increasing levels of cyclinD1 and retinoblastoma protein; as a result NSC proliferation was doubled. Finally, BRCA-1 is expressed while NSCs proliferate, but is drastically down regulated upon differentiation, indicating that BRCA-1 could be used as a possible NSC marker. In summary, in this thesis estrogen and nandrolone were identified as NSC regulators which decrease proliferation and positively influence neurogenesis. Also, we have identified the hormone α-MSH as a NSC mitogen, and BRCA-1 as a possible NSC marker.

Spinal cord injury (SCI) is a devastating condition characterized by the loss of neuronal pathways responsible for coordinating motor and sensory information between the brain and the rest of the body. The mammalian spinal cord is limited in its ability to repair itself, so treatments devised to replace damaged tissue and promote regeneration are essential towards developing a cure. This work describes the development of a guidance channel strategy for spinal cord transection. Chitosan guidance channels were designed as a delivery vehicle for neural stem/progenitor cell (NSPC) transplants and drug-eluting poly(lactic-co-glycolic acid) (PLGA) microspheres. PLGA microspheres were embedded into chitosan channels by a spin-coating method. These microsphere-loaded channels demonstrated the ability for controlled short-term bioactive release of the small molecule drug dibutyryl cyclic-AMP (dbcAMP) and long-term bioactive release of the protein alkaline phosphatase. NSPCs were shown to be responsive to dbcAMP delivery, which results in greatly enhanced differentiation into neurons. The effect of directed neuronal differentiation was investigated after spinal cord transection in rat, resulting in a dramatic increase in NSPC transplant survival. Guidance channels containing NSPCs treated with dbcAMP resulted in robust tissue bridge formation after SCI, demonstrating extensive axonal regeneration and promoting functional recovery.

Spinal cord injury (SCI) is a devastating condition characterized by the loss of neuronal pathways responsible for coordinating motor and sensory information between the brain and the rest of the body. The mammalian spinal cord is limited in its ability to repair itself, so treatments devised to replace damaged tissue and promote regeneration are essential towards developing a cure. This work describes the development of a guidance channel strategy for spinal cord transection. Chitosan guidance channels were designed as a delivery vehicle for neural stem/progenitor cell (NSPC) transplants and drug-eluting poly(lactic-co-glycolic acid) (PLGA) microspheres. PLGA microspheres were embedded into chitosan channels by a spin-coating method. These microsphere-loaded channels demonstrated the ability for controlled short-term bioactive release of the small molecule drug dibutyryl cyclic-AMP (dbcAMP) and long-term bioactive release of the protein alkaline phosphatase. NSPCs were shown to be responsive to dbcAMP delivery, which results in greatly enhanced differentiation into neurons. The effect of directed neuronal differentiation was investigated after spinal cord transection in rat, resulting in a dramatic increase in NSPC transplant survival. Guidance channels containing NSPCs treated with dbcAMP resulted in robust tissue bridge formation after SCI, demonstrating extensive axonal regeneration and promoting functional recovery.

Neural stem cell self-renewal and differentiation are tightly regulated events during CNS development, leading to cell division into new neural stem cells or the formation of neurons and glial cells. This thesis focuses on the cellular responses induced by known and novel factors in neural stem and progenitor cells (NSPCs). Platelet-derived growth factor (PDGF) signaling has previously been implicated in NSPC regulation as well as in tumor formation. In order to evaluate the differentiation process and find new regulators of NSPCs a micro-array screen was performed, evaluating transcription during normal differentiation and the effect of PDGF-AA in this process. The transcriptional profile of PDGF-AA treated NSPCs was shown to be an intermediate between the profiles of neural stem cells and their progeny. The NSPC transcriptome was also found to have similarities with that of experimental glioma. A previously non-characterized transcript, the nuclear receptor binding protein 2 (NRBP2), was identified and found to be expressed in the developing and adult mouse brain and in medulloblastoma. NRBP2 down-regulation rendered neural progenitors sensitive to induced cell death. Different PDGF ligands interact with different combinations of PDGF receptors. Therefore NSPCs were stimulated with either PDGF-AA or -BB to further evaluate cellular responses with regard to the two specific isoforms. A divergent effect between the two isoforms in long-term proliferation and cell survival was found, with PDGF-BB being the most efficient stimulator. Stem cell factor (SCF) has previously been identified as a regulator in the hematopoietic system and we showed that SCF induces a migratory response in NSPCs. In addition, SCF positively affected cell survival but had no effect on NSPC differentiation. Insights into the regulatory mechanisms involved in neural stem cell signaling are needed to develop diagnostic tools and novel treatments.

The classical cell cycle regulatory pathway is well appreciated as a key regulator of cell fate determination during neurogenesis; however, the extent of pRB/E2F function in neural stem and progenitor cells is not fully understood, and insight into the mechanisms underlying its connection with cell fate regulation are lacking. The E2F3 transcription factor has emerged as an important regulator of neural precursor cell (NPC) proliferation in the embryonic and adult forebrain, and we demonstrate here that it also influences the self-renewal potential of NPCs. Using knockout mouse models of individual E2F3 isoforms, we demonstrate the surprising result that the classical transcriptional activator E2F3a represses NPC self-renewal and promotes neuronal differentiation, while E2F3b promotes the expansion of the NPC pool and inhibits differentiation. We attribute these opposing activities to a unique mechanism of transcriptional regulation at the Sox2 locus, a key regulator of stem cell pluripotency, whereby E2F3a recruits transcriptional repressors to this site, and E2F3b promotes Sox2 activation. Importantly, E2F3a-mediated Sox2 regulation is necessary for cognitive function in the adult. Additionally, through the determination of genome-wide promoter binding sites for E2f3 isoforms as well as E2F4, another key regulator of NPC self-renewal, we determined that E2Fs are poised to regulate an extensive set of target genes with key roles in regulating diverse cell fate choices in NPCs, including self-renewal, cell death, progenitor expansion, maintenance of the precursor state, and differentiation. Together, these results reveal a diversity of function for E2Fs in the control of neural precursor cell fate, and identify E2F3 isoforms as important regulators of the pluripotency and stem cell maintenance gene Sox2.

abstract: Modern medical conditions, including cancer, traumatic brain injury, and cardiovascular disease, have elicited the need for cell therapies. The ability to non-invasively track cells in vivo in order to evaluate these therapies and explore cell dynamics is necessary. Magnetic Resonance Imaging provides a platform to track cells as a non-invasive modality with superior resolution and soft tissue contrast. A new methodology for cellular labeling and imaging uses Nile Red doped hexamethyldisiloxane (HMDSO) nanoemulsions as dual modality (Magnetic Resonance Imaging/Fluorescence), dual-functional (oximetry/ detection) nanoprobes. While Gadolinium chelates and super paramagnetic iron oxide-based particles have historically provided contrast enhancement in MRI, newer agents offer additional advantages. A technique using 1H MRI in conjunction with an oxygen reporter molecule is one tool capable of providing these benefits, and can be used in neural progenitor cell and cancer cell studies. Proton Imaging of Siloxanes to Map Tissue Oxygenation Levels (PISTOL) provides the ability to track the polydimethylsiloxane (PDMS) labeled cells utilizing the duality of the nanoemulsions. 1H MRI based labeling of neural stem cells and cancer cells was successfully demonstrated. Additionally, fluorescence labeling of the nanoprobes provided validation of the MRI data and could prove useful for quick in vivo verification and ex vivo validation for future studies. / Dissertation/Thesis / Masters Thesis Bioengineering 2014