Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 117-135.

Huntington’s disease (HD) is a fatal neurodegenerative disease caused by a CAG repeat expansion in exon 1 of the huntingtin gene, resulting in an expanded polyglutamine (polyQ) repeat in the huntingtin protein. Patients receive symptomatic treatment for motor, emotional, and cognitive impairments; however, there is no treatment to slow the progression of the disease, with death occurring 15-20 years after diagnosis. Mutant huntingtin protein interferes with multiple cellular processes leading to cellular dysfunction and neuronal loss. Due to the complexity of mutant huntingtin toxicity, many approaches to treating each effect are being investigated. Unfortunately, addressing one cause of toxicity might not result in protection from other toxic insults, necessitating a combination of treatments for HD patients. Ideally, single therapy targeting the mutant mRNA or protein could prevent all downstream toxicities caused by mutant huntingtin. In this work, I used animal models to investigate a potential therapeutic target for decreasing mutant huntingtin protein, and I apply bioluminescent imaging to investigate RNA interference to silence mutant huntingtin target sites. The enzyme puromycin sensitive aminopeptidase (PSA) has the unique property of degrading polyQ peptides and been implicated in the degradation of huntingtin. In this study, we looked for an effect of decreased PSA on the pathology and behavior in a mouse model of Huntington’s disease. To achieve this, we crossed HD mice with mice with one functional PSA allele and one inactivated PSA allele. We found that PSA heterozygous HD mice develop a greater number of pathological inclusion bodies, representing an accumulation of mutant huntingtin in neurons. PSA heterozygous HD mice also exhibit worsened performance on the raised-beam test, a test for balance and coordination indicating that the PSA heterozygosity impairs the function of neurons with mutant huntingtin. In order to test whether increasing PSA expression ameliorates the HD phenotype in mice we created an adeno-associated virus (AAV) expressing the human form of PSA (AAV-hPSA). Unexpectedly, testing of AAV-hPSA in non-HD mice resulted in widespread toxicity at high doses. These findings suggest that overexpression of PSA is toxic to neurons in the conditions tested. In the second part of my dissertation work, I designed a model for following the silencing of huntingtin sequences in the brain. Firefly luciferase is a bioluminescent enzyme that is extensively used as a reporter molecule to follow biological processes in vivo using bioluminescent imaging (BLI). I created an AAV expressing the luciferase gene containing huntingtin sequences in the 3'-untranslated region (AAV-Luc-Htt). After co-injection of AAV-Luc-Htt with RNA-silencing molecules (RNAi) into the brain, we followed luciferase activity. Using this method, we tested cholesterol-conjugated siRNA, un-conjugated siRNA, and hairpin RNA targeting both luciferase and huntingtin sequences. Despite being able to detect silencing on isolated days, we were unable to detect sustained silencing, which had been reported in similar studies in tissues other than the brain. We observed an interesting finding that co-injection of cholesterol-conjugated siRNA with AAV-Luc-Htt increased luminescence, findings that were verified in cell culture to be independent of serotype, siRNA sequence, and cell type. That cc-siRNA affects the expression of AAV-Luc-Htt reveals an interesting interaction possibly resulting in increased delivery of AAV into cells or an increase in luciferase expression within the cell. My work presents a method to follow gene silencing of huntingtin targets in the brain, which needs further optimization in order to detect sustained silencing. Finally, in this dissertation I continue the study of bioluminescent imaging in the brain. We use mice that have been injected in the brain with AAV-Luciferase (AAV-Luc) to screen 34 luciferase substrate solutions to identify the greatest light-emitting substrate in the brain. We identify two substrates, CycLuc1 and iPr-amide as substrates with enhanced light-emitting properties compared with D-luciferin, the standard, commercially available substrate. CycLuc1 and iPr-amide were tested in transgenic mice expressing luciferase in dopaminergic neurons. These novel substrates produced luminescence unlike the standard substrate, D-luciferin which was undetectable. This demonstrates that CycLuc1 and iPr-amide improve the sensitivity of BLI in low expression models. We then used CycLuc1 to test silencing of luciferase in the brain using AAV-shRNA (AAV-shLuc). We were unable to detect silencing in treated mice, despite a 50% reduction of luciferase mRNA. The results from this experiment identify luciferase substrates that can be used to image transgenic mice expressing luciferase in dopaminergic neurons. My work contributes new data on the study of PSA as a modifier of Huntington’s disease in a knock-in mouse model of Huntington’s disease. My work also makes contributions to the field of bioluminescent imaging by identifying and testing luciferase substrates in the brain to detect low level of luciferase expression.

Neuronal nicotinic acetylcholine receptors (nAChRs) are involved in a plethora of fundamental biological processes ranging from muscle contraction to the formation of memories. The studies described in this work focus on the transcriptional regulation of the CHRNB4 gene, which encodes the ß4 subunit of neuronal nAChRs. We previously identified a regulatory sequence (5´– CCACCCCT –3´), or “CA box”, critical for CHRNB4 promoter activity in vitro. Here I report transcription factor interaction at the CA box along with an in vivo analysis of CA box transcriptional activity. My data indicate that Sp1, Sp3, Sox10 and c-Jun interact with the CHRNB4 CA box in the context of native chromatin. Using an in vivo transgenic approach in mice, I demonstrated that a 2.3-kb fragment of the CHRNB4 promoter region, containing the CA box, is capable of directing cell-type specific expression of a reporter gene to many of the brain regions that endogenously express the CHRNB4 gene. Site-directed mutagenesis was used to test the hypothesis that the CA box is critical for CHRNB4 promoter activity in vivo. Transgenic animals were generated in which LacZ expression is driven by a mutant form of the CA box. Reporter gene expression was not detected in any tissue or cell type at ED18.5. Similarly, I observed dramatically reduced reporter gene expression at PD30 when compared to wild type transgenic animals, indicating that the CA box is an important regulatory feature of the CHRNB4 promoter. ChIP analysis of brain tissue from mutant transgenic animals demonstrated that CA box mutation results in decreased interaction of the transcription factor Sp1 with the CHRNB4 promoter. I have also investigated transcription factor interaction at the CHRNB4 promoter CT box, (5´– ACCCTCCCCTCCCCTGTAA –3´) and demonstrated that hnRNP K interacts with the CHRNB4 promoter in an olfactory bulb derived cell line. Surprisingly, siRNA experiments demonstrated that hnRNP K knockdown has no impact on CHRNA5, CHRNA3 or CHRNB4 gene expression. Interestingly, knockdown of the transcription factor Purα results in significant decreases in CHRNA5, CHRNA3 and CHRNB4 mRNA levels. These data indicate that Purα can act to enhance expression of the clustered CHRNA5, CHRNA3 and CHRNB4 genes. Together, these results contribute to a more thorough understanding of the transcriptional regulatory mechanisms underlying expression of the CHRNB4 as well as the CHRNA5 and CHRNA3 genes, critical components of cholinergic signal transduction pathways in the nervous system.

No description available.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from PDF title page (viewed on Sept. 17, 2009). Includes bibliographical references.

Synaptic plasticity, the ability of synapses to change in strength, underlies complex brain functions such as learning and memory, yet little is known about the precise molecular mechanisms and downstream signaling pathways involved. The major goal of my doctoral thesis was to understand these molecular mechanisms and cellular processes underlying synaptic plasticity using the Drosophilalarval neuromuscular junction (NMJ) as a model system. My work centered on a signaling pathway, the Wg/Wnt signaling pathway, which was found to be crucial for activity-driven synapse formation. The Wg/Wnt family of secreted proteins, besides its well-characterized roles in embryonic patterning, cell growth and cancer, is beginning to be recognized as a pivotal player during synaptic differentiation and plasticity in the brain. At the DrosophilaNMJ, the Wnt-1 homolog Wingless (Wg) is secreted from presynaptic terminals and binds to Frizzled-2 (DFz2) receptors in the postsynaptic muscle. Perturbations in Wg signaling lead to poorly differentiated NMJs, containing synaptic sites that lack both neurotransmitter release sites and postsynaptic structures. In collaboration with other members of the Budnik lab, I set out to unravel the mechanisms by which Wg regulates synapse differentiation. We identified a novel transduction pathway that provides communication between the postsynaptic membrane and the nucleus, and which is responsible for proper synapse development. In this novel Frizzled Nuclear Import (FNI) pathway, the DFz2 receptor is internalized and transported towards the nucleus. The C-terminus of DFz2 is subsequently cleaved and imported into the postsynaptic nucleus for potential transcriptional regulation of synapse development (Mathews, Ataman, et al. Science (2005) 310:1344). My studies also centered on the genetic analysis of Glutamate Receptor (GluR) Interacting Protein (dGRIP), which in mammals has been suggested to regulate the localization of GluRs and more recently, synapse development. I generated mutations in the gene, transgenic strains carrying a dGRIP-RNAi and fluorescently tagged dGRIP, and antibodies against the protein. Remarkably, I found dgrip mutants had synaptic phenotypes that closely resembled those in mutations altering the FNI pathway. Through the genetic analysis of dgrip and components of the FNI pathway, immunoprecipitation studies, electron microscopy, in vivotrafficking assays, time-lapse imaging, and yeast two-hybrid assays, I demonstrated that dGRIP had a hitherto unknown role as an essential component of the FNI pathway. dGRIP was found in trafficking vesicles that contain internalized DFz2. Further, DFz2 and dGRIP likely interact directly. Through the use of pulse chase experiments I found that dGRIP is required for the transport of DFz2 from the synapse to the nucleus. These studies thus provided a molecular mechanism by which the Wnt receptor, DFz2, is trafficked from the postsynaptic membrane to the nucleus during synapse development and implicated dGRIP as an essential component of the FNI pathway (Ataman et al. PNAS (2006) 103:7841). In the final part of my dissertation, I concentrated on understanding the mechanisms by which neuronal activity regulates synapse formation, and the role of the Wnt pathway in this process. I found that acute changes in patterned activity lead to rapid modifications in synaptic structure and function, resulting in the formation of undifferentiated synaptic sites and to the potentiation of spontaneous neurotransmitter release. I also found that these rapid modifications required a bidirectional Wg transduction pathway. Evoked activity induced Wg release from synaptic sites, which stimulated both the postsynaptic FNI pathway, as well as an alternative presynaptic Wg pathway involving GSK-3ß/Shaggy. I suggest that the concurrent activation of these alternative pathways by the same ligand is employed as a mechanism for the simultaneous and coordinated assembly of the pre- and postsynaptic apparatus during activity-dependent synapse remodeling (Ataman et al. Neuron (2008) in press). In summary, my thesis work identified and characterized a previously unrecognized synaptic Wg/Wnt transduction pathway. Further, it established a mechanistic link between activity-dependent synaptic plasticity and bidirectional Wg/Wnt signaling. These findings provide novel mechanistic insight into synaptic plasticity.

Lung cancer is the leading cause of cancer-related mortality worldwide. The main risk factor associated with lung cancer is cigarette smoking. Research through the years suggests that nicotine in cigarettes promotes lung cancer by activating signaling pathways that lead to cell proliferation, cell survival, angiogenesis, and metastasis. Nicotine’s cellular actions are mediated by its cognate receptors, nicotinic acetylcholine receptors (nAChRs). Here, I describe the expression levels of all known human nAChR subunit genes in both normal and lung cancer cells. Of note, the genes encoding the α5, α3, and β4 subunits (CHRNA5/A3/B4) are over-expressed in small cell lung carcinoma (SCLC), the most aggressive form of lung cancer. This over-expression is regulated by ASCL1, a transcription factor important in normal lung development and lung carcinogenesis. The CHRNA5/A3/B4 locus has recently been the focus of a series of genetic studies showing that polymorphisms in this region confer risk for both nicotine dependence and lung cancer. I show that CHRNA5/A3/B4 depletion results in decreased SCLC cell viability. Furthermore, while nicotine promotes SCLC cell viability and tumor growth, blockade of α3β4 nAChRs inhibits SCLC cell viability. These results suggest that increased expression and function of nAChRs, specifically the α3β4α5 subtype, potentiate the effects of nicotine in SCLC. This dual hit from the carcinogens in tobacco and the cancer-promoting effects of nicotine, may provide a possible mechanism for the increased aggressiveness of SCLC. In addition, nAChRs can be activated by the endogenous ligand, acetylcholine, which acts as an autocrine/paracrine growth factor in SCLC. Increased function of α3β4α5 nAChRs in SCLC could also potentiate acetylcholine’s mitogenic effects. This mechanism, combined with other known autocrine/paracrine growth loops in SCLC, may help explain the ineffectiveness of available therapies against SCLC. In an effort to add to the current arsenal against SCLC, I screened a 1280-compund library using a bioluminescence-based viability assay I developed for high-throughput applications. Primary screening, followed by secondary and tertiary verification, indicate that pharmacologically active compounds targeting neuroendocrine markers inhibit SCLC cell viability.

A common feature of many neuropathies is axon degeneration. While the reasons for degeneration differ greatly, the process of degeneration itself is similar in most cases. Axon degeneration after axotomy is termed ‘Wallerian degeneration,’ whereby injured axons rapidly fragment and disappear after a short period of latency (Waller, 1850). Wallerian degeneration was thought to be a passive process until the discovery of the Wallerian degeneration slow (Wlds) mouse mutant. In these mice, axons survive and function for weeks after nerve transection. Furthermore, when the full-length protein is inserted into mouse models of disease with an axon degeneration phenotype (such as progressive motor neuronopathy), Wlds is able to delay disease onset (for a review, see Coleman, 2005). Wlds has been cloned and was found to be a fusion event of two neighboring genes: Ube4b, which encodes an ubiquitinating enzyme, and NMNAT-1 (nicotinamide mononucleotide adenylyltransferase-1), which encodes a key factor in NAD (nicotinamide adenine dinucleotide) biosynthesis, joined by a 54 nucleotide linker span (Mack et al., 2001). To address the role of Wlds domains in axon protection and to characterize the subcellular localization of Wlds in neurons, our lab developed a novel method to study Wallerian degeneration in Drosophila in vivo (MacDonald et al., 2006). Using this method, we have discovered that mouse Wlds can also protect Drosophila axons for weeks after acute injury, indicating that the molecular mechanisms of Wallerian degeneration are well conserved between mouse and Drosophila. This observation allows us to use an easily manipulated genetic model to move the Wlds field forward; we can readily identify what Wlds domains give the greatest protection after injury and where in the neuron protection occurs. In chapter two of this thesis, I identify the minimal domains of Wlds that are needed for protection of severed Drosophila axons: the first 16 amino acids of Ube4b fused to Nmnat1. Although Nmnat1 and Wlds are nuclear proteins, we find evidence of a non-nuclear role in axonal protection in that a mitochondrial protein, Nmnat3, protects axons as well as Wlds. In chapter 3, I further explore a role for mitochondria in Wlds-mediated severed axon protection and find the first cell biological changes seen in a Wlds-expressing neuron. The mitochondria of Wlds- and Nmnat3-expressing neurons are more motile before injury. We find this motility is necessary for protection as suppressing the motility with miro heterozygous alleles suppresses Wldsmediated axon protection. We also find that Wlds- and Nmnat3- expressing neurons show a decrease in calcium fluorescent reporter, gCaMP3, signal after axotomy. We propose a model whereby Wlds, through production of NAD in the mitochondria, leads to an increase in calcium buffering capacity, which would decrease the amount of calcium in the cytosol, allowing for more motile mitochondria. In the case of injury, the high calcium signal is buffered more quickly and so cannot signal for the axon to die. Finally, in chapter 4 of my thesis, I identify a gene in an EMS-based forward genetic screen which can suppress Wallerian degeneration. This mutant is a loss of function, which, for the first time, definitively demonstrates that Wallerian degeneration is an active process. The mammalian homologue of the gene encodes a mitochondrial protein, which in light of the rest of the work in this thesis, highlights the importance of mitochondria in neuronal health and disease. In conclusion, the work presented in this thesis highlights a role for mitochondria in both Wlds-mediated axon protection and Wallerian degeneration itself. I identified the first cell biological changes seen in Wlds-expressing neurons and show that at least one of these is necessary for its protection of severed axons. I also helped find the first Wallerian degeneration loss-of-function mutant, showing Wallerian degeneration is an active process, mediated by a molecularly distinct axonal degeneration pathway. The future of the axon degeneration field should focus on the mitochondria as a potential therapeutic target.

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A proteína miostatina, é um regulador negativo do crescimento muscular e a modulação de sua expressão pode consistir em tratamento para distrofias musculares. Nós estudamos expressão endógena da miostatina no músculos gastrocnêmio e diafragma de 4 modelos murinos de degeneração muscular: os camundongos Dmdmdx, SJL/J, Largemyd e Lama2dy-2J/J. Observamos que a miostatina é menos expressa no músculo gastrocnêmio do que diafragma normal, refletindo um músculo mais sujeito a lesão. Nas quatro linhagens distróficas a miostatina é menos expressa do que em camundongos normais, tanto no músculo gastrocnêmio como diafragma, sem diferença entre os dois. A analise comparativa da degeneração e regeneração muscular mostrou maior correlação da inibição da miostatina com o padrão de degeneração. Nossos resultados sugerem que o processo de degeneração, quando iniciado, e independentemente de seu grau, causa molecular primária, ou músculo afetado, parece atuar de forma similar na inibição da expressão da miostatina, possivelmente como estimulo a regeneração do dano. / Myostatin is a negative regulator of muscle growth, and its inhibition has been considered a therapeutic strategy for muscular dystrophies. We evaluated the endogenous expression of myostatin in the gastrocnemius and diaphragm muscles from 4 mouse dystrophic models including Dmdmdx, SJL/J>, Largemyd and Lama2dy2J/J. In normal mice, we observed that myostatin is less expressed in the gastrocnemius than in the diaphragm, reflecting a muscle most prone to lesions. In the 4 dystrophic models, myostatin expression was reduced, in both gastrocnemius and diaphragm muscles. The comparative analysis of the histopathology of the muscles with the expression of myostatin showed a stronger correlation with the pattern of degeneration then regeneration. Our results suggest that, when started, the process of degeneration of the muscle, independently of the primary molecular defect, or degree, seems to act in a similar pathway leading to the inhibition of the expression of myostatin in the affected muscles, possibly as a stimulus to regeneration of damage.