In the development of the central nervous system, thousands of neuronal subtypes must be generated, each with their own unique molecular properties. This process is governed by selector transcription factors, which specify cell-identities by binding to cell-type specific genomic regulatory elements. These regulatory elements, dispersed across extragenic regions of the genome, establish precise long-distance interactions with target gene promoters to regulate their expression. While prior studies have emphasized the roles of distally bound selector transcription factors in cell-type specification, the involvement of gene promoters in the regulation of gene expression remains underexplored. In this dissertation, I analyze the role of promoter elements in regulating neuronal gene expression programs using a comprehensive approach that combines high-throughput genomics and targeted experimental manipulations. In Chapter 2, I reveal a highly flexible regulatory system utilized in the nervous system: neuronal promoters are universal and can thus be activated by any enhancer found within their regulatory neighborhood. This model of promiscuous neuronal promoters raises two important questions: Is promoter promiscuity a universal phenomena? What are the promoter elements that facilitate universality? To address these questions in Chapter 3, I first find that promoters of genes associated with pluripotency exhibit incompatibility with neuronal enhancers. Then, to test what promoter elements encode for this incompatibility, and also which elements endow neuronal promoters with their promiscuity, I developed a novel promoter-screening strategy. Through this work, I discovered novel aspects of enhancer-promoter communication. First, core promoters are universal and can be induced by non-cell identity matched distal enhancers. Second, promoter-proximal regions serve to modulate expression from universal core promoters by either dampening or potentiating their responsiveness to distal enhancers. This work suggests that in addition to distal regulatory elements, promoter-proximal regions also play an active role in fine-tuning cell-type specific gene expression programs by either modulating induction or repressing ectopic expression. Finally, in Chapter 4, I explore another aspect of the regulation of cell-identity during development, shifting my focus away from selector transcription factors and instead on “secondary” transcription factors induced during differentiation. Here, I utilize a multi-omic approach to characterize the role of Mnx1 in motor neuron development. Analysis of its effects on gene expression, distal genomic binding patterns, and influence on the overall regulatory landscape reveals that Mnx1 plays a role in maintaining the motor neuron cell-identity by ensuring robust expression of motor neuron genes and preventing ectopic expression of genes normally restricted to alternate neuronal subtypes. This suggests that “secondary” transcription factors play a role in refining cellular identities established by selector transcription factors. Integrating these findings with prior research in central nervous system development underscores that while neuronal gene expression programs are primarily established through the actions of selector transcription factor-bound distal regulatory elements, promoters and secondary transcription factors contribute to the fine-tuning of transcription and, consequently, cell identity.

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Neurovascular injuries are leading causes of disability implicated in neurological dysfunction. Much of the Central Nervous System (CNS) homeostasis depends on concerted signaling between neurons, glial cells, and vasculature–the neurovascular unit (NVU). Neurovascular injuries disrupt the NVU causing hypoxia, ischemia, neuroinflammation, and neuronal death. Much of the neuroinflammatory responses associated with neurovascular injuries have been characterized, but the contribution of specific signaling pathways from the injured endothelium to inflammatory response remains to be established. To understand vascular-glial communication in the context of vascular injury, the Troy lab has used a mouse model of retinal vascular injury, retinal vein occlusion (RVO). The retina is a CNS enclosed tissue that allows live visualization of vascular and neuronal condition upon injury, genotype, and/or treatment. Previous studies in the laboratory determined that non-apoptotic expression of endothelial caspase-9 (EC Casp9) was key for the development of retinal edema, capillary ischemia, and neuronal death. Caspases are known for their role in mediating cell death, but how and if glial cells orchestrated outcomes remain unknown. This thesis work aimed to investigate the role of caspase-9 signaling in vascular-glial communication and its contribution to pro-inflammatory cytokine levels and neurodegeneration in neurovascular injury. To answer this, we first optimized the mouse model of RVO and profiled the levels of caspases in RVO retinas treated or untreated with a caspase-9 inhibitor using immunohistochemistry. Then, we used tamoxifen inducible endothelial and astroglial caspase-9 KO lines, subjected them to RVO and measured glial changes, cytokine levels, capillary ischemia, retinal edema, neuronal death, and vision dysfunction. We first found that RVO induces a range of cell-specific levels of caspases and that inhibition of caspase-9 specifically modulated the levels of endothelial caspase-9 and 8, neuronal caspase-9, 7, and 6, astroglial caspase-6, and leukocytic caspase-9 and 7. Our studies also suggest that endothelial caspase-9 induces a decrease in reactive microglia, inflammatory cytokines, cleaved- caspase-6 and GFAP cleavage in astrocytes. EC Casp9 deletion also altered changes in GFAP, nestin and AQP4 levels in Müller glia. Through investigating an astroglial caspase-9 KO, we discovered that astroglial caspase-9 could be upstream of astroglia caspase-6. Additionally, we found that astroglial caspase-9 loss protected hypoxic retinas from capillary ischemia but not from retinal edema nor neuronal death. Lastly, we used an optokinetic test to study the potential role of endothelial and astroglial caspase-9 in RVO-induced vision disfunction. Our results indicate that removing caspase-9 from endothelial cells or astrocytes protected contrast sensitivity damage in visual function one day post-RVO. In sum, the present thesis work demonstrates that endothelial and astroglial caspase-9 signaling can lead to inflammation and worsening of visual function in neurovascular injury.

Treatment for neurological diseases has been limited by the presence of the protective blood-brain barrier. Recent studies from our laboratory have shown that direct intraventricular implantation of microcapsules containing genetically modified cells can effectively deliver the transgene product to the mouse brain, thereby circumventing the blood-brain barrier. In this thesis, the experiments were aimed at scaling up the murine experiments to determine if direct implantation of alginate-poly-L-lysine-alginate microcapsules to the central nervous system of dogs was a feasible means of treating the large animal brain. In the first two experiments reported here, microcapsules containing cells genetically modified to secrete human growth hormone were injected into the central nervous system of dogs. Two routes of delivery were examined, intraventricular brain surgery and injection into the spinal intrathecal space (cisterna magna). While empty capsules within the central nervous system were benign, microcapsules containing cells induced an acute inflammatory response in the brain and spinal cord tissue, irrespective of the route of delivery. Human growth hormone was detected transiently in four of six dogs, but the data were interpreted with caution due to extraneous variables such as compromised microcapsules in two of the dogs and previous systemic treatment in six of the other dogs. In the last experiment, microcapsules containing cells genetically modified to secrete the lysosomal enzyme a-L-iduronidase were implanted into the lateral ventricles of a dog with Mucopolysaccharidosis type I in an attempt to correct the characteristic neuronal pathology. An immune response ensued and appeared to abolish any possible effect of the microcapsule treatment. The experiments presented here demonstrate the challenges and obstacles that need to be overcome to effectively scale up therapies from rodent experiments to large animals. The data also shed light on the immunological complications that may arise with invasive and repeated treatment in the central nervous system of large animals. / Thesis / Master of Science (MS)

(Abstract shortened by UMI.) / The conclusion of the studies reported herein can be summarized as follows: (1) PI in TCD correlates well with WMC volume and helps to differentiate those with and without WMC in stroke patients. (2) Post-stroke cognitive complaints are not related to severity of WMC among lacunar stroke patients. (3) The ARWMC scale correlates with objective cognitive performances and the operational definitions of ARWMC scale improves inter-rater reliability on CT. (4) Cognitive impairment in patients with confluent WMC is mediated by global and frontal cortical atrophy. Predictors for cognitive progression are cortical atrophy, absence of hyperlipidemia, low BP, and low cognitive scores. / With an aging population, prevalence of dementia is expected to escalate in the coming decades. The burden is especially great in developing countries like China. Similar to Alzheimer's pathology (e.g. amyloid plaque), age-related white matter changes (WMC) are important substrates of dementia. Since WMC are considered to be of ischemic origin, dementia related to WMC is believed to be more preventable than Alzheimer's disease. Yet, studies focusing on WMC have been relatively few. The thesis will cover 4 aspects of WMC and cognitive impairment. / Xiong, Yunyun. / Adviser: Vincent Mok. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 198-244). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese; some appendixes in Chinese.

The main theme of this thesis is that there is a common structural basis for drugs acting on the central nervous system (CNS), and that this concept may be used to design new CNS-active drugs which have greater specificity and hence less side-effects. To develop these ideas, the biological basis of how drugs modify CMS neurotransmission is described, and illustrated using dopaminergic pathways. An account is then given of the use of physicochemical concepts in contemporary drug design. The complete conformational analysis of several antipsychotic drugs is used to illustrate some of these techniques in the development of a model for antipsychotic drug action. After reviewing current structure-activity studies in several classes of CNS drugs (antipsychotics, anti-depressants, stimulants, hal1ucinogens, anticonvulsants and analgesics), a hypothesis for a common structural basis of CNS drug action is proposed- This is based on a topographical comparison of the X-ray structures of eight representative CNS-active drugs, and consists of three parts: 1.there is a common structural basis for the activity of many different CNS-active drug classes; 2. an aromatic ring and a nitrogen atom are the primary binding groups whose topographical arrangement is fundamental to the activity of these drug classes; 3. the nature and placement of secondary binding determines different classes of CNS drug activity. A four-Point model for this common structural basis is then defined using 14- CNS-active drug structures that include the original eight used in proposing the hypothesis. The coordinates of this model are: R1 (0. 3.5, 0), R2 (0, -3.5, O), N (4.8. -0.3, 1.4), and R3 (6.3, 1.3, 0), where R1 and R2 represent the point locations of a hydrophobic interaction of the common aromatic ring with a receptor, and R3 locates the receptor point for a hydrogen bond involving the common nitrogen, N. Extended structures were used to define the receptor points R1, R2 and R3, and the complete conformational space of each of the 14 molecules was considered. It is then shoun that the model may be used to predict whether a given structure is likely to show CNS activity: a search over 1,000 entries in the current Merck Index shows a high probability (82%) of CNS activity in compounds fitting the structural model. Analysis of CNS neurotransmitters and neuropeptides shows that these fit the common model well. Based on the available evidence supporting chemical evolution, protein evolution, and the evolution of neurotransmitter functions, it is surmised that the aromatic ring/nitrogen atom pharmacophore proposed in the common model supports the idea of the evolution of CNS receptors and their neurotransmitters, possibly from an aromatic amine or acety1cho1ine acting as a primaeval communicating molecule. The third point in the hypothesis trilogy is then addressed. The extensive conformation-activity analyses that have resulted in well-defined models for five separate CNS drug classes are used to map out the locations of secondary binding groups relative to the common model for anti-psychotics, antidepressants, analgesics, anticholinergics, and anticonvulsants. With this information, and knowledge derived from receptor-binding data, it is postulated that drugs having specified activity could be designed. In order to generate novel structures having a high probability of CNS-activity, a process of drug design is described in which known CNS structures are superimposed topographically using the common model as a template. Atoms regarded as superfluous may be selectively deleted and the required secondary binding groups added in predicted locations to give novel structures. It is concluded that this process provides the basis for the rational design of new lead compounds which could further be optimized for potent and specific CNS activity.