Theses and Dissertations

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Open Access
Dihydrotestosterone attenuates endotoxin, cytokine, and hypoxia-induced vascular inflammation
(Colorado State University. Libraries, 2011) Osterlund, Kristen Leanne, author; Handa, Robert, advisor; Gonzales, Rayna, committee member; Amberg, Gregory, committee member; Garrity, Deborah, committee member; Tobet, Stuart, committee member
Vascular inflammation plays a key role in the etiology of cardiovascular disease, particularly stoke. Vascular inflammation is under the control of several transcription factors, including nuclear factor kappa B and hypoxia inducible factor-1 alpha (HIF-1α). Activation of these transcription factors can lead to the production of inflammatory mediators such as cyclooxygenase-2 (COX-2). COX-2 plays a role in vascular inflammation, cerebral ischemia-induced injury, and has been implicated as a source of reactive oxygen species (ROS). Inflammatory mediators, such as endotoxin or cellular breakdown products released following injury, are known to signal through the Toll-like receptor 4 (TLR4). TLR4 activation leads to NFκB activation and subsequent production of COX-2. Like COX-2, TLR4 has also been implicated in injury-induced oxidative stress and cerebral ischemia damage. Previous studies have demonstrated that gonadal steroid hormones can also modulate vascular inflammation. Both protective and detrimental effects of androgens on the cardiovascular system have been reported. Since the potent androgen receptor (AR) agonist dihydrotestosterone (DHT) can be converted to 3β-diol, an estrogen receptor (ER) β-selective agonist, I hypothesized that ERβ may mediate some of the protective effects of androgens, while the AR may mediate some of the detrimental effects. The overall goal of this dissertation was to determine the mechanisms by which androgens can influence the vascular inflammatory response under both physiological and pathophysiological conditions. The hypothesis to be tested was that DHT influences vascular inflammation under both physiological and pathophysiological conditions. In my first set of experiments, using Western blot, I found that DHT increases expression of the vascular inflammatory mediator COX-2 under physiological conditions in human coronary artery vascular smooth muscle (VSM) cells and human brain VSM cells. This effect of DHT was attenuated in the presence of the AR antagonist bicalutamide. This data indicates that the pro-inflammatory effect of DHT under normal physiological conditions is AR mediated. In my second set of experiments, I examined the effects of DHT on vascular inflammation under a variety of pathophysiological conditions. Surprisingly, I found that DHT decreased cytokine-induced COX-2 expression and oxidative stress, endotoxin-induced COX-2 and TLR4 expression in human VSM cells. Furthermore, DHT also decreased hypoxia induced HIF-1α and COX-2 expression in human brain VSM cells and rat pial arteries. Finally, I found that DHT decreased hypoxia with glucose deprivation (HGD)-induced HIF-1α, COX-2 and TLR4 expression in human brain VSM cells. DHT`s anti-inflammatory effects during cytokine or HGD-induced inflammation in human brain VSM cells were not blocked by the AR antagonist bicalutamide, indicating that they were not AR mediated. These results led me to my second hypothesis, that DHT's anti-inflammatory effects are ERβ-mediated. In my third set of experiments, I found that the DHT metabolite/ERβ selective agonist 3β-diol also decreased cytokine-induced COX-2 expression in human brain VSM cells. Furthermore, DHT's ability to reduce cytokine-induced COX-2 expression in human brain VSM cells was inhibited by the non-selective estrogen receptor antagonist ICI 182,780 and the selective ERβ antagonist PHTPP. The mRNAs for steroid metabolizing enzymes in the pathway necessary to convert DHT to 3β-diol were detected in human brain VSM cells, as were AR and ERβ mRNAs. Therefore, DHT appears to be protective against cerebrovascular inflammation via conversion to 3β-diol and subsequent activation of ERβ in human brain VSM cells. The results of these studies indicate that: 1) DHT increases COX-2 expression under unstimulated/physiological conditions via an AR-dependent mechanism. 2) DHT decreases cytokine-, endotoxin,-hypoxia, and HGD-induced COX-2 expression via an AR-independent mechanism. 3) DHT decreases cytokine-induced reactive oxygen species. 4) DHT decreases hypoxia-induced HIF-1α expression. 5) DHT decreases HIF-1α and TLR4 expression during HGD via an AR-independent mechanism. 6) DHT's effect to attenuate cytokine-induced COX-2 expression is ERβ-mediated.
Open Access
Electrophysiological analysis of Kv2 channel regulation by non-canonical and canonical mechanisms
(Colorado State University. Libraries, 2020) Maverick, Emily E., author; Tamkun, Michael, advisor; Amberg, Gregory, committee member; Krapf, Diego, committee member; Tsunoda, Susan, committee member; Vigh, Jozsef, committee member
Kv2 channels are the most abundant voltage-gated potassium channels in the mammalian nervous system and entire body. These channels regulate action potential ﬁring and apoptosis via their canonical conducting functions. However, Kv2 channels also play a non-conducting role in the cells in which they are expressed. Speciﬁcally, they form junctions between the endoplasmic reticulum and plasma membranes, and these junctions regulate a myriad of cellular process. Several studies have now shown that many Kv2.1 channels expressed on the plasma membranes of mammalian cells do not respond canonically to changes in membrane voltage. Instead of opening to allow potassium efﬂux, the pores of these non-canonical channels are locked in a non-conducting state. This state has likely evolved to prevent electrical paralysis that would otherwise be conferred upon cells expressing high levels of completely functional Kv2 channels. The mechanism bringing about the non-conducting state of Kv2.1 channels is unknown. The work described in the ﬁrst part of this dissertation was carried out with the ultimate goal of revealing the mechanism of the Kv2.1 channel non-conducting state. I describe an improved, all-electrophysiological method to quantify the numbers of nonconducting Kv channels expressed in heterologous systems. I validate this approach by measuring the fraction of non-conducting Kv2.1 channels that arise when expressed in HEK293 cells. I go on to use this approach to show evidence for a non-conducting state in the second Kv2 isoform, Kv2.2, for the ﬁrst time. I ﬁnd that like Kv2.1, the Kv2.2 nonconducting state is dependent on the density of channels in the membrane. Surprisingly, I also ﬁnd that two Shaker-related channels, Kv1.4 and Kv1.5 also show density dependence in the fraction of channels that conduct. These results suggest that the mechanism underlying the non-conducting state is more common than we thought, and I discuss hypotheses that should be tested in the future. In the last part of this dissertation I describe the effects of the assembly of Kv2 channels with a newly discovered family of Kv β subunits, the AMIGOs. The experiments in this portion of the dissertation focus on each AMIGO's ability to modulate canonical, conducting Kv2 channels, as well as Kv2's ability to alter AMIGO trafﬁcking and localization. I ﬁnd that both Kv2.1 and Kv2.2 promote AMIGO trafﬁcking to the plasma membrane and alter their localization there. I also ﬁnd that while all three AMIGO isoforms promote Kv2 channel opening, AMIGO2 confers an additional stabilizing effect on the open state by slowing inactivation and deactivation. In all, the work in this dissertation expands on our current understanding of Kv channel function. These ﬁndings should guide future experiments to probe both canonical and non-canonical functions of Kv channels.
Embargo
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.
Open Access
Molecular mechanisms regulating Kv2.1-induction of endoplasmic reticulum / plasma membrane contact sites
(Colorado State University. Libraries, 2019) Johnson, Ben, author; Tamkun, Michael, advisor; Amberg, Gregory, committee member; Di Pietro, Santiago, committee member; Prenni, Jessica, committee member; Tsunoda, Susan, committee member
Kv2 voltage gated potassium channels localize to 'clusters' on the soma, axon initial segment, and dendritic arbor of hippocampal neurons. For decades the molecular mechanism behind this localization pattern was unknown. In 2015 our lab determined that this behavior was due to the channels interacting with an unknown endoplasmic reticulum resident protein and thereby forming endoplasmic reticulum / plasma membrane (ER/PM) junctions. The channel clusters covering the surface of cells represented those domains. The work in this dissertation examines in increased detail the mechanism, regulation, and possible functions associated with these sites. ER/PM junctions are domains with a variety of roles. They regulate both calcium and lipid homeostasis, they are involved in vesicular trafficking, and they oversee a host of cell signaling pathways. Junctions represent 12% of the neuronal soma surface and are also present in both the axon and the dendritic arbor. These are sites that exhibit a high degree of dynamic flux, both in composition and in structure. Residency of junction proteins is governed by the calcium concentration of the ER, the calcium concentration of the cytosol, the activity of the excitable cell, and the lipid composition of the PM. In turn these residents influence the nature of the junction, determining the function and nanoarchitecture of these domains. In this work we use a proximity-based biotinylation approach to identify VAMP-associated proteins (VAPs) as the Kv2 channel interactor responsible for the formation of ER/PM junctions. We characterize the amino acid motif necessary to generate interaction between the two proteins, finding an unconventional FFAT motif located in the channel C-terminus. We examine the protein composition of these novel junctions by investigating their relationship with other known ER/PM tethers such as Nir2, STIM1 and the junctophilins. We use super resolution imaging techniques to observe ER membrane behavior at these locations and study how that behavior changes during the concentration of additional protein residents. Lastly, we investigate the mechanisms underlying Kv2-VAP junction disassembly during neuronal activity and insult. We find that Kv2.1-VAP unbinding during glutamate stimulation is mediated by serine residues downstream of the Kv2.1 FFAT motif. This dispersal of Kv2-VAP ER/PM junctions during calcium influx is mirrored by junctophilin-induced junction disassembly, suggesting a common mechanism regulating ER/PM junctions throughout the hippocampus. This dissertation examines a novel microdomain formed by Kv2 channels and presents data describing how this domain is created and regulated on a molecular level. It represents the first in-depth study of this topic.
Open Access
Sexual divergence in prefrontal neural regulation and encoding of depression-associated behaviors
(Colorado State University. Libraries, 2022) Wallace, Tyler, author; Myers, Brent, advisor; Hentges, Shane T., advisor; Amberg, Gregory, committee member; Conner, Bradley, committee member
Major depressive disorder (MDD) accounts for the most years lived with disability worldwide. Yet, despite its staggering prevalence, the biological mechanisms underpinning MDD onset are not understood, further complicated by considerable sex-based differences in MDD occurrence. The ventromedial prefrontal cortex (vmPFC) is heavily associated with MDD, though how vmPFC neural populations respond to and regulate behaviors associated with MDD, including affective state, social behaviors, and stress responding is unknown. Thus, I utilized viral methods to dissect how a genetically identified neural population within the vmPFC regulates and encodes MDD-associated behaviors. In chapter 2, I utilized an optogenetic technique to increase the firing rate of a subset of glutamatergic vmPFC neurons in conjunction with behavioral testing. My results demonstrated considerable sexual divergence in vmPFC glutamatergic influence. In males, stimulation conferred positive affect, increased social motivation, and constrained aspects of the acute stress response. While in females, stimulation did not alter behavior and augmented the acute stress response. In chapter 3, I utilized a similar optogenetic technique to dissect how vmPFC projections to the posterior hypothalamus (PH), contribute to behavioral and physiological regulation. Again, my results demonstrated sexual divergence in vmPFC circuit function. In males, stimulation of the vmPFC to PH glutamatergic circuit conferred positive affect, and constrained aspects of the acute stress response, though it did not alter social behavior. The circuit similarly conferred positive affect in females, but again augmented the acute stress response. Overall, my stimulation of vmPFC glutamatergic neurons identified that they regulate affect, social behavior, and stress responding but the specific effects are sex and circuit specific. While chapters 2 and 3 identified how specific vmPFC neural populations can regulate behavioral and physiological processes, how these neural populations respond to behavior and how these responses are disrupted in pathology was unknown. Thus, in chapter 4, I utilized fluorescent calcium indicators to record the activity of genetically-identified vmPFC glutamatergic neurons during behavioral testing. To determine changes to vmPFC neural activity in pathology, animals were exposed to a preclinical model of MDD, chronic variable stress. My results showed that vmPFC glutamatergic neurons are responsive to object, social, stressful, and rewarding stimuli regardless of sex. However chronic stress exposure altered vmPFC glutamatergic activity in males more so than females, with some of these differences accounted for by female ovarian status. Overall, the work presented in this dissertation determined how a vmPFC neural population regulates MDD-disrupted behaviors, detailed how a specific vmPFC circuit contributes to this regulatory role and measured how vmPFC neurons respond to behavior in real-time with and without a history of chronic stress.
Open Access
The physiological function and pathological significance of transient receptor potential ankyrin 1 channels in the cerebral artery endothelium
(Colorado State University. Libraries, 2015) Sullivan, Michelle Nicole, author; Earley, Scott, advisor; Feng, Yumei, advisor; Dinenno, Frank, committee member; Tjalkens, Ronald, committee member; Amberg, Gregory, committee member
Endothelial cell Ca2+ dynamics have a significant influence on cerebrovascular tone. Several transient receptor potential (TRP) channels have been shown to mediate Ca2+ influx in the endothelium, including TRP vanilloid 4 (TRPV4), TRPV3, and TRP ankyrin 1 (TRPA1), which activates endothelium-dependent vasodilatory pathways. High resolution Ca2+ techniques have allowed for the recording of unitary TRP channel Ca2+ influx events, called TRP sparklets, in endothelial cells where they have been found to underlie vascular function. The following studies first characterize the biophysical properties of TRPV4 and TRPA1 sparklets in endothelial cells. TRPA1 channels are present in the endothelium of cerebral arteries and absent from other vascular beds, suggesting a critical, yet previously unknown function for the channel in this tissue. Research here describes the physiological function of TRPA1 channels as sensors of oxidative membrane degradation in cerebral artery endothelial cells. Further, the involvement of TRPA1 channels in delaying the onset of hypertension-associated spontaneous hemorrhagic stroke is examined.

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