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We are interested in understanding the molecular mechanisms that govern synaptic growth and plasticity. Recent evidence from several laboratories suggests that small GTPases play an important role in the promotion of neurite outgrowth; however, their role in the control of synaptic growth and functional plasticity is not well understood. The goal of this thesis is to investigate the role of small GTPases (including Rac, Rho and Cdc42) in the regulation of synaptic growth in vivo, using the Drosophila larval neuromuscular junction (NMJ) synapses as a model system. Our results show that presynaptic overexpression of Rac, but not of Rho or Cdc42, positively regulates both synaptic structure and function. Genetic loss of Rac leads to embryonic lethality, making it impossible to assess the full loss-of-function phenotype using conventional mutants. To circumvent this, we use the MARCM (Mosaic Analysis with a Repressible Cell Marker) technique to generate single motor neuron clones devoid of all genetic copies of Rac. Our data suggest that Rac activity is crucial for normal synaptic development. In support of this conclusion, we demonstrate that genetic removal of trio, a guanine nucleotide exchange factor (GEF) that is known to activate Rac, leads to a drastic reduction in the number of synaptic boutons. In addition, genetic removal of one copy of trio is sufficient to suppress the gain-of-function phenotype of Rac. Moreover, we demonstrate that partial removal of the fragile X mental retardation gene (dfmr1), a known suppressor of Rac, enhances the gain-of-function phenotype of Rac. Taken together, our findings support a model in which Rac signaling positively regulates synaptic growth and function at the Drosophila larval NMJ.

Diss. (sammanfattning) Umeå : Univ., 2009. / Härtill 4 uppsatser.

Thesis (Ph.D. in Human Medical Genetics) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 117-126). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D. in Cell and Developmental Biology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 139-155).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 88-101).

Axonal and synaptic degeneration is a hallmark of peripheral neuropathy, brain injury, and neurodegenerative disease. Axonal degeneration has been proposed to be mediated by an active autodestruction program, akin to apoptotic cell death; however, loss-of-function mutations capable of potently blocking axon self-destruction have not been described. Using a forward genetic screen in Drosophila, we identified that loss of the Toll receptor adaptor dSarm (sterile a/Armadillo/Toll-Interleukin receptor homology domain protein) cell-autonomously suppresses Wallerian degeneration for weeks after axotomy. Severed mouse Sarm1 null axons exhibit remarkable long-term survival both in vivo and in vitro, indicating that Sarm1 prodegenerative signaling is conserved in mammals. Our results provide direct evidence that axons actively promote their own destruction after injury and identify dSarm/Sarm1 as a member of an ancient axon death signaling pathway. This death signaling pathway can be activated without injury by loss of the N-terminal self-inhibitory domain, resulting in spontaneous neurodegeneration. To investigate the role of axon self-destruction in disease, we assessed the effects of Sarm1 loss on neurodegeneration in the SOD1-G93A model of amyotrophic lateral sclerosis (ALS), a lethal condition resulting in progressive motor neuron death and paralysis. Loss of Sarm1 potently protects motor axons and synapses from degeneration, but only extends animal survival by 10%. Thus, there appears to be at least two driving forces in place during ALS disease progression: (1) Sarm1 mediated axon death, and (2) cell body destruction via some unknown mechanism.

Every fall, Northeastern America monarch butterflies (Danaus plexippus) undergo an extraordinary migration to their overwintering site in Central Mexico. During their long migration, monarch migrants use sun compass to navigate. To maintain a southward flying direction, monarch migrants compensate for the continuously changing position of the sun by providing timing information to the compass using their circadian clock. Animal circadian clocks depend primarily on a negative transcriptional feedback loop to track time. I started my work to re-construct the monarch butterfly circadian clock negative feedback loop in cell culture, focusing on homologs of Drosophila clock genes. It turned out that in addition to a Drosophila-like cryptochrome (cry1) gene, a second mammalian-like cry2 gene exists in monarch butterflies and many other insects, except in Drosophila. The two CRYs showed distinct functions in our initial assays in cultured Drosophila Schneider 2 (S2) cells. CRY2 functions as a potent transcriptional repressor, while CRY1 is light sensitive but shows no obvious transcriptional activity. The existence of two cry genes in insects changed the Drosophila-centric view of insect circadian clock. During the course of my study, our lab obtained a monarch cell line called DpN1 cells. These cells possess a light-driven clock and contributed tremendously to the research on monarch circadian clock. Using this cell line, I provided strong evidence supporting monarch CRY2’s role as a major circadian clock repressor and identified a protein-protein protective interaction cascade underlying the CRY1-mediated resetting of the molecular oscillator in DpN1 cells. I continued my work trying to understand how insect CRY2 inhibits transcription. I provided evidence suggesting the involvement of monarch PER in promoting CRY2 nuclear entry in both S2 cells and DpN1 cells. Finally, I mapped CRY2’s transcriptional inhibitory activity onto its N-terminal domain. Collectively, my research helped to change our view of insect clocks from a Drosophila-centric standpoint to a much more diverse picture. My studies also advanced the understanding of monarch circadian clock mechanism, and provides a foundation for further studies.

The work in presented in this thesis identifies Drosophila Wnt8 as a novel zygotic target of the Dorsal/Twist/Snail network using a microarray analysis to identify differentially expressed genes in maternal dorsal mutant and gain-of-function Toll10b embryos as compared to wild type. In-situ hybridization with a Wnt8 antisense RNA probe revealed a fairly complex expression pattern in the early embryo. No maternal expression was observed and the first zygotic expression appeared at stage 4 at the poles. This was followed by patchy and relatively weak expression in the presumptive mesoderm with stronger expression in the mesectoderm and later the neuroectoderm. These expression required the Dorsal/Twist/Snail network with some input from Delta in the neuroectoderm. All embryonic Wnt8 expression ceased after late stage 10. Snail was found to repress Wnt8 in the presumptive mesoderm. The relevance of Wnt8 as a Snail target was tested by bypassing this repression in wild type embryos using a maternal Gal4 driver to drive UAS-Wnt8. This led to a loss of ventral furrow formation and a phenocopy of the snail mutant phenotype, thereby indicating that the repression of Wnt8 by Snail is important for gastrulation. Further investigation into the mechanism revealed a reduction in the expression of multiple target genes of Dorsal (including snail) in these Wnt8 overexpressing embryos. Ventral nuclear Dorsal protein was reduced as compared to wild type, suggesting that high levels of Wnt8 can antagonize Dorsal nuclear localization. Deficiency embryos lacking Wnt8 showed the opposite phenotype of expanded anterior and posterior snail RNA staining, as well as an expanded nuclear Dorsal signal in the posterior. This could be phenocopied using dsRNA against Wnt8, and was fully rescueable in the deficiency background using a Wnt8 genomic fragment. It has been reported that loss-of-function snail embryos lose the sharp lateral boundaries and high levels of snail expression more rapidly as compared to wild type. We hypothesize that this loss is due to derepressed Wnt8 antagonizing Dorsal and consequently its target, snail. In support of our hypothesis, double mutants of snail and the Wnt8 deficiency show a rescue of the snail pattern, though not a rescue of ventral furrow formation. Western blot analysis reveals a decrease in the levels of phosphorylation of Dorsal in Wnt8 overexpressing embryos as compared to wild type. Phosphorylation of Dorsal is required for its nuclear translocation. Hence, these data corroborate the observation of reduced nuclear Dorsal in embryos overexpressing Wnt8. Together, these data point to Wnt8 being an important target and a feedback inhibitor of the Dorsal/Twist/Snail pathway that achieves its effect by the inhibition of Dorsal.

Vesicular trafficking, the movement of vesicles between organelles and the plasma membrane for secretion, consists of multiple highly regulated processes. Many protein families function as specificity and regulatory determinants to ensure correct vesicle targeting and timing of trafficking events. The SNARE proteins dock and fuse vesicles to their target membranes. Sec1/Munc18 (SM) proteins regulate membrane fusion through interactions with the SNAREs—SM proteins have been shown to act as both inhibitors and stimulators of SNARE assembly and membrane fusion. However, the details of these SM protein functions are not understood. Constructing a model of SM protein function has been challenging due to the various modes of interactions reported between SM proteins and their SNAREs. SM proteins interact with their cognate SNAREs and SNARE complexes through several distinct modes. The most conserved mode is an interaction with the syntaxin N-peptide; other modes of binding, such as the syntaxin closed conformation, are hypothesized to be specific for specialized cell types. In order to elucidate the general function of SM proteins, I investigated the function of the endosomal SM protein Vps45p by analyzing its interactions with its cognate syntaxin Tlg2p and its role in SNARE assembly. I had two main hypotheses: that the Tlg2p N-peptide does not solely mediate the interaction between Vps45p and Tlg2p; and that Vps45p functions to stimulate SNARE complex assembly. I systematically mapped the interaction between Vps45p and Tlg2p using various Tlg2p truncations containing the different domains of Tlg2p and discovered a second binding site on Tlg2p that corresponds to the closed conformation. The neuronal SM-syntaxin pair interacts in a similar manner, indicating that this interaction mode is conserved. To characterize the closed conformation binding mode further, and determine its relationship to the N-peptide binding mode, I developed a quantitative fluorescent electrophoretic mobility shift assay. Results indicate that these two sites do not bind simultaneously and that the N-peptide binding modulates the closed conformation affinity. Furthermore, I monitored the effect of Vps45p on SNARE complex assembly using size exclusion chromatography. Under the conditions tested, Vps45p did not appear to stimulate SNARE complex assembly. The work presented here addresses several puzzling issues in the field and significantly contributes to the construction of a new mechanistic model for SM protein function. In this new model, the SM protein is recruited to the membrane by its interaction with the syntaxin N-peptide. The SM protein then binds the syntaxin closed conformation thus inhibiting SNARE complex assembly. Upon dissociation of the SM protein from the closed conformation, an event perhaps regulated by the SM protein, syntaxin opens and interacts with the other SNAREs to form a SNARE complex. Fusion ensues, stimulated by the SM protein.