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Browsing by Author "Hoerndli, Frederic, committee member"

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    An investigation of synaptic vesicle docking and priming and a proposed method for quantitatively measuring both in Drosophila using electron tomography
    (Colorado State University. Libraries, 2023) Twiggs, Jasmin A., author; Reist, Noreen, advisor; Hoerndli, Frederic, committee member; Hoke, Kim, committee member; Tamkun, Michael, committee member
    The nervous system, as the body's command center, plays a crucial role in cellular communication within the brain and between the brain and other body systems. Neurons, the individual cellular units, transmit electrical information and communicate with other cells through neurotransmitter release in response to electrical stimuli. Chapter 1 introduces the foundational concepts of neuronal structure and function and delves into the mechanisms underlying neurotransmitter release. Special attention is given to the neuromuscular junction (NMJ), a well-studied chemical synapse crucial for muscle movement. The synaptic vesicle cycle is introduced, with particular emphasis on docking and priming. The significance of active zones, specialized sites for efficient signal transmission, and their associated structural components are underscored. Synaptotagmin, a pivotal protein in calcium-triggered vesicle fusion, is discussed with emphasis on its C2B polylysine motif. Throughout the chapter, the utility of Drosophila as a model system for studying synaptic processes, particularly at the NMJ, is emphasized. In sum, Chapter 1 provides the foundational knowledge essential for comprehending the intricate cellular and molecular facets of synaptic communication within the nervous system, serving as a precursor to subsequent chapters' investigations. Chapter 2 examines synaptotagmin's C2B polylysine motif and its role in synaptic vesicle docking at the Drosophila NMJ. It explores the polylysine motif's potential involvement in endocytosis, demonstrates an unaffected interaction with AP-2, and uses electron microscopy to find no significant changes in vesicle distribution. The findings suggest that the reduced neurotransmitter release in the polylysine mutant is likely due to an impairment in vesicle priming. Chapter 3 introduces a method for studying synaptic vesicle docking and priming in Drosophila, using electron tomography. I address the limitations of conventional electron microscopy and underscore the need for higher-resolution techniques to assess molecular structures that mediate physiological processes. Chapter 3 also emphasizes the significance of the contact area between docked vesicles and the presynaptic membrane as a correlate of vesicle priming. The protocol, expected results, and key considerations are discussed. The methods presented in Chapter 3 offer a promising approach for understanding synaptic processes. In Chapter 4, I discuss key considerations for when standard electron microscopy can be used for assessing vesicle docking. Then, I discuss how the electron tomography method presented in Chapter 3 could not only confirm the results found in Chapter 2, that the synaptotagmin C2B polylysine motif is not implicated in vesicle docking but could also be used to directly test the mutant's role in priming. Specific aims for future studies on the synaptotagmin polylysine mutation in Drosophila are presented, potential results and interpretations are discussed. Finally, I showcase interesting, unpublished findings from electron tomograms I have taken at the Drosophila NMJ and discuss their potential significance.
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    Gas6/AXL signaling contributes to GnRH-dependent activation of pituitary gonadotropes
    (Colorado State University. Libraries, 2023) Mohammad Zadeh, Pardis, author; Amberg, Gregory C., advisor; Hoerndli, Frederic, committee member; Stasevich, Timothy, committee member; Winger, Quinton, committee member
    Gonadotropin-releasing hormone (GnRH) receptor plays a fundamental role in reproduction and is prevalent in various urogenital, reproductive, and non-reproductive cancers. Beyond the conventional G protein-coupled receptor signaling, GnRH receptors interact functionally with multiple receptor tyrosine kinases. AXL, a receptor tyrosine kinase found in various tissues and numerous tumors, is the focus of this dissertation to discover its impact, along with its endogenous ligand Gas6, on GnRH receptor signaling. In this study, clonal murine pituitary αT3-1 and LβT2 gonadotrope cell lines were utilized to evaluate the effect of AXL activation on GnRH receptor-dependent signaling pathways. A combination of ELISA and immunofluorescence techniques was employed to analyze AXL and GnRH receptor expression in αT3-1 and LβT2 cells, as well as in murine and human pituitary sections. Additionally, ELISA was used to quantify alterations in ERK phosphorylation, pro-MMP9 production, and the release of LHβ. The abundance of Egr-1 transcripts was measured using digital droplet PCR. To assess αT3-1 and LβT2 cell migration responses to GnRH and AXL, trans-well migration assay was used. Results showed the presence of AXL, alongside GnRH receptors, in αT3-1 and LβT2 gonadotrope cell lines, as well as in murine and human pituitary sections. In line with AXL's potentiating role, Gas6 enhanced GnRH-dependent ERK phosphorylation in αT3-1 and LβT2 cells. Furthermore, Gas6 increased the abundance of Egr-1 transcripts, suggesting enhanced post-transcriptional GnRH receptor responses. Notably, in LβT2 cells, Gas6/AXL signaling not only stimulated LHβ production but also enhanced GnRH receptor dependent pro-MMP9 protein generation and promoted cell migration, underlining its functional significance. In summary, our findings unveil a hitherto undiscovered role for AXL as a modulator of GnRH receptor signaling.
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    miR-137 regulates PTP61F, affecting insulin signaling, metabolic homeostasis, and starvation resistance in Drosophila melanogaster
    (Colorado State University. Libraries, 2023) Saedi, Hana Ibrahim, author; Tsunoda, Susan, advisor; Hoerndli, Frederic, committee member; Amberg, Gregory, committee member; Di Pietro, Santiago, committee member
    miR-137 is a highly conserved brain-enriched microRNA (miRNA) that has been associated with neuronal function and proliferation. Here, we show that Drosophila miR-137 null mutants display increased body weight with enhanced triglyceride and glucose levels and decreased locomotor activity. When challenged by nutrient deprivation, miR-137 mutants exhibit reduced motivation to feed and significantly prolonged survival. Together, these phenotypes suggest a new role for miR-137 in energy homeostasis. Genetic epistasis experiments show that the starvation resistance of miR-137 mutants involves the insulin signaling pathway, and that loss of miR-137 results in drastically reduced phosphorylation/activation of the single insulin receptor, InR, in Drosophila. We explore the possibility that the protein tyrosine phosphatase61F (PTP61F), ortholog of TC-PTP/PTP1B, known to dephosphorylate InR across species, is a potential in vivo target of miR-137. We show that loss of miR-137 results in upregulation of an endogenously tagged PTP61F protein, and that genetically increasing levels of PTP61F mimics the loss of phosphorylated InR and increased starvation resistance seen in miR-137 mutants. Finally, we show that the enhanced starvation resistance of miR-137 mutants is normalized by activation of the insulin signaling pathway in the nervous system. Our study introduces miR-137 as a new player in the regulation of central insulin signaling and metabolic homeostasis.
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    Na+ -activated K+ channels protect against overexcitation and seizure-like behavior in Drosophila
    (Colorado State University. Libraries, 2021) Byers, Nathan S., author; Tsunoda, Susan, advisor; Garrity, Deborah, committee member; Hentges, Shane, committee member; Hoerndli, Frederic, committee member; Tamkun, Michael, committee member
    Na+-activated K+ channels (KNa) encode K+ channels that are activated by internal Na+ and are widely expressed throughout the mammalian central nervous system. Based on the biophysical properties of the channels, it has long been postulated that they act as a reserve mechanism to combat neuronal overexcitation. Specifically, early electrophysiological recordings suggested that only when intracellular Na+ levels rise significantly, for instance in neuropathological conditions, do KNa channels become active. More recent evidence suggests that they may function under normal physiological circumstances by means of binding cytoplasmic factors and via the persistent Na+ current. However, to date it is unclear if KNa channels function to prevent overexcitation in vivo. Therefore, research in my dissertation sets out to test the hypothesis that KNa channels protect against overexcitation in Drosophila models of epilepsy. Drosophila contain one gene encoding a KNa channel, dSlo2. In the third chapter of this dissertation, I examine expression of dSlo2 channels throughout the nervous system. Findings from this chapter show that dSlo2 channels are expressed in cholinergic neurons, the main excitatory neuron of the Drosophila brain. Furthermore, dSlo2 channels were excluded from GABAergic neurons. I additionally found that dSlo2 channels are localized to axonal regions of multiple neuronal subtypes in the nervous system. Thus, these results suggest that as K+ channels widely and preferentially expressed in excitatory neurons in the brain, dSlo2 channels may function to dampen neuronal, and perhaps behavioral, excitability. In Chapter 4, I test the hypothesis that dSlo2 channels protect against behavioral abnormalities caused by cholinergic overexcitation. I first show that the loss of dSlo2 exacerbates behavioral deficits and death associated with prolonged exposure to a cholinergic agonist, Imidacloprid. Furthermore, I found that adult flies lacking dSlo2 exhibit mechanically induced seizure-like behavior following feeding of Imidacloprid, which does not occur in wild-type flies. Combined, these results suggest that dSlo2 channels do indeed protect against cholinergic overexcitation. It has previously been shown that mammalian KNa channels are activated by a persistent Na+ current (INaP) in neurons, suggesting that these channels may ameliorate behavioral consequences of an increased INaP in vivo. In Chapter 5, I test the hypothesis that dSlo2 channels protect against Drosophila seizure-like behavior induced by an increased INaP. I find that the loss of dSlo2 significantly exacerbates seizure-like behavior in multiple Drosophila epileptic models, including a model for human generalized epilepsy with febrile seizures plus (GEFS+). Additionally, the absence of dSlo2 worsens seizure-like behavior when flies are exposed to Veratridine, a pharmacological agent known to increase INaP. Interestingly, the loss of dSlo2 also revealed a spontaneous seizure phenotype in INaP-affected seizure models that was otherwise absent. Altogether, these results are consistent with the model that KNa channels are activated by INaP, and protect against seizure-like behavior actuated by increased INaP. Overall, the work in my dissertation expands our understanding of the role of KNa channels. These findings suggest that KNa channels may play a protective role for many neuropathological diseases associated with an increased INaP, such as epilepsy, amyotrophic lateral sclerosis, neuropathic pain, and ischemia.
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    The discovery of novel proteins regulating melanosome biogenesis and function
    (Colorado State University. Libraries, 2022) Detry, Anna, author; Di Pietro, Santiago, advisor; Hansen, Jeffrey, committee member; Hoerndli, Frederic, committee member
    Melanosomes are lysosomal related organelles found in cells which are responsible for making pigment such as skin melanocytes. They are membrane bound organelles that form from the endosomal pathway and have specific proteins and enzymes which allow them to perform the function of melanin production. The process of melanosome biogenesis involves the melanosomes developing through four stages that are classified by electron microscopy appearance. The different melanosome stages have amyloid fibrils formed by proteolytically processed PMEL protein, different amounts of melanin, and different melanosomal proteins. In addition to melanosome biogenesis, another key factor in proper melanin formation and pigmentation is the melanosome luminal pH. The melanin producing enzyme tyrosinase is a pH dependent enzyme. When melanosomes are more acidic, tyrosinase is less functional, leading to less melanin production and a hypopigmentation phenotype. The Di Pietro lab and others have shown that the Two Pore Channel Two (TPC2) is a key regulator of melanosome pH, as well as a regulator of melanosome size and localizes to melanosome membranes. A proximity-dependent biotin identification experiment was preformed using TPC2 and eight potential melanosome proteins were identified. Each of these candidate proteins were knocked down in a human melanoma cell line using small interfering RNA and studied for a potential pigmentation phenotype. Tetraspanin10, phospholipase D1, myosin heavy chain 9, and myosin heavy chain 10 all showed a hypopigmentation phenotype. Two independent tetraspanin 10 knockout cell lines were generated using CRISPR-Cas9 which reproduced the hypopigmentation phenotype. In addition, the phenotype was rescued by re-expressing tetraspanin 10 in the knockout cells and overexpressing tetraspanin 10 in wild type cells showed a hyperpigmentation phenotype. This shows that tetraspanin 10 is involved in the pigmentation process. CD63 is another tetraspanin known to play vital roles in melanosome biogenesis and based on the minimal information aviable on tetraspanin 10, it can be hypothesized as being involved in PMEL processing. The discovery that tetraspanin 10 is involved in skin pigmentation will lead to better understanding of the pigmentation process and pigmentation related diseases.